U.S. patent application number 17/608242 was filed with the patent office on 2022-08-04 for vaginal microbiome markers for prediction of prevention of preterm birth and other adverse pregnancy outcomes.
The applicant listed for this patent is VIRGINIA COMMONWEALTH UNIVERSITY. Invention is credited to Tom ARODZ, Paul BROOKS, Gregory BUCK, David EDWARDS, Jennifer FETTWEIS, Philippe GIRERD, Kimberly K. JEFFERSON, Myrna SERRANO, Jerome STRAUSS.
Application Number | 20220243247 17/608242 |
Document ID | / |
Family ID | |
Filed Date | 2022-08-04 |
United States Patent
Application |
20220243247 |
Kind Code |
A1 |
ARODZ; Tom ; et al. |
August 4, 2022 |
VAGINAL MICROBIOME MARKERS FOR PREDICTION OF PREVENTION OF PRETERM
BIRTH AND OTHER ADVERSE PREGNANCY OUTCOMES
Abstract
A method for determining the risk of an adverse pregnancy
outcome for a woman is provided comprising the steps of measuring a
level of TM7-H1 and optionally one or more of BVAB1, Sneathia
amnii, and Prevotella cluster 2 in a vaginal sample obtained from
the woman, and identifying the woman as having an increased risk
for an adverse pregnancy outcome, or other adverse pregnancy
outcomes, if the levels are increased compared to corresponding
standard control levels. Methods for the prophylactic treatment of
subjects identified as being at increased risk for an adverse
pregnancy outcome are also provided.
Inventors: |
ARODZ; Tom; (Richmond,
VA) ; BUCK; Gregory; (Richmond, VA) ;
JEFFERSON; Kimberly K.; (Richmond, VA) ; STRAUSS;
Jerome; (Richmond, VA) ; FETTWEIS; Jennifer;
(Richmond, VA) ; SERRANO; Myrna; (Richmond,
VA) ; GIRERD; Philippe; (Richmond, VA) ;
BROOKS; Paul; (Richmond, VA) ; EDWARDS; David;
(Richmond, VA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VIRGINIA COMMONWEALTH UNIVERSITY |
Richmond |
VA |
US |
|
|
Appl. No.: |
17/608242 |
Filed: |
May 1, 2020 |
PCT Filed: |
May 1, 2020 |
PCT NO: |
PCT/US2020/030934 |
371 Date: |
November 2, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62842724 |
May 3, 2019 |
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International
Class: |
C12Q 1/06 20060101
C12Q001/06; C12Q 1/689 20060101 C12Q001/689 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under grant
number R01 HD080784 awarded by the National Institutes of Health
(NIH). The government has certain rights in the invention.
Claims
1. A method for determining the risk of an adverse pregnancy
outcome for a woman, comprising: measuring an abundance of
Saccharibacteria TM7-H1 and optionally one or more of BVAB1,
Sneathia amnii, and Prevotella cluster 2 in a vaginal sample
obtained from the woman, and identifying the woman as having an
increased risk for an adverse pregnancy outcome if the abundance is
increased compared to corresponding standard control levels.
2. The method of claim 1, wherein the abundance of TM7-H1, BVAB1,
Sneathia amnii, and Prevotella cluster 2 is measured.
3. The method of claim 1, further comprising measuring an abundance
of one or more of: Dialister cluster 51, Prevotella amnii, Sneathia
sanguinegens, Aerococcus christensenii, Clostridales BVAB2,
Coriobacteriaceae OTU27, Dialister micraerophilus, Parvimonas
OTU142, Megasphaera OTU70 type 1, and Lactobacillus crispatus
cluster.
4. The method of claim 1, wherein the adverse pregnancy outcome is
preterm birth (PTB).
5. The method of claim 1, wherein the woman is pregnant and the
vaginal sample is obtained between 6 and 24 weeks of gestation.
6. The method of claim 1, wherein the woman is pregnant and the
vaginal sample is obtained between 1 and 6 weeks of gestation.
7. The method of claim 1, wherein the vaginal sample is obtained
prior to pregnancy.
8. The method of claim 1, wherein the woman is of African
ancestry.
9. A method for determining the risk of an adverse pregnancy
outcome for a woman and administering at least one prophylactic
treatment to a woman identified as being at risk for an adverse
pregnancy outcome, comprising: i) measuring an abundance of TM7-H1
and optionally one or more of BVAB1, Sneathia amnii, and Prevotella
cluster 2 in a vaginal sample obtained from the woman; ii)
identifying the woman as having an increased risk for an adverse
pregnancy outcome if the abundance is increased compared to
corresponding standard control levels; and iii) administering the
at least one prophylactic treatment for the adverse pregnancy
outcome to the woman who is identified as having an increased risk
for the adverse pregnancy outcome.
10. The method of claim 9, wherein the abundance of TM7-H1, BVAB1,
Sneathia amnii, and Prevotella cluster 2 is measured.
11. The method of claim 9, further comprising measuring an
abundance of one or more of: Dialister cluster 51, Prevotella
amnii, Sneathia sanguinegens, Aerococcus christensenii,
Clostridales BVAB2, Coriobacteriaceae OTU27, Dialister
micraerophilus, Parvimonas OTU142, Megasphaera OTU70 type 1, and
Lactobacillus crispatus cluster.
12. The method of claim 9, wherein the adverse pregnancy outcome is
PTB.
13. The method of claim 9, wherein the woman is pregnant and the
vaginal sample is obtained between 6 and 24 weeks of gestation.
14. The method of claim 9, wherein the woman is pregnant and the
vaginal sample is obtained between 1 and 6 weeks of gestation.
15. The method of claim 9, wherein the vaginal sample is obtained
prior to pregnancy.
16. The method of claim 9, wherein the at least one PTB
prophylactic treatment is selected from the group consisting of
antenatal corticosteroids, antibiotics, tocolytics, progesterone,
cerclage application, products that modify the conditions of the
female reproductive tract, prebiotics, probiotics, genetically
engineered microbial or bacteriophage preparations, vaginal
microbial transplants, and combinations thereof.
17. The method of claim 9, wherein the woman is of African
ancestry.
18. A method of detecting an adverse pregnancy outcome risk,
comprising: obtaining a vaginal sample from a pregnant woman at a
gestation of 6-24 weeks; and detecting an abundance of TM7-H1,
BVAB1, Sneathia amnii, and Prevotella cluster 2 in the vaginal
sample.
19. A method of detecting an adverse pregnancy outcome risk,
comprising: obtaining a vaginal sample from a pregnant woman at a
gestation of 1-6 weeks; and detecting an abundance of TM7-H1,
BVAB1, Sneathia amnii, and Prevotella cluster 2 in the vaginal
sample.
20. A method of detecting an adverse pregnancy outcome risk,
comprising: obtaining a vaginal sample from a woman prior to
pregnancy; and detecting an abundance of TM7-H1, BVAB1, Sneathia
amnii, and Prevotella cluster 2 in the vaginal sample.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/842,724, filed May 3, 2019, which is hereby
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0003] The invention is generally related to vaginal microbiome
signatures that are indicative of a higher risk for adverse
pregnancy outcomes and the use of such signatures for the
prevention of adverse pregnancy outcomes such as preterm birth,
miscarriage, or preeclampsia.
BACKGROUND OF THE INVENTION
[0004] The incidence of preterm birth, with its significant
societal costs, remains above 10% worldwide. Approximately 15
million preterm births defined as those that occur before 37 weeks
of gestation occur annually worldwide.sup.1. Preterm birth (PTB)
remains the second most common cause of neonatal death across the
globe, and the most common cause of infant mortality in countries
with middle and high income economies.sup.1,2. The consequences of
PTB persist from early childhood.sup.34 into adolescence.sup.5-7
and adulthood.sup.8-10. In the US, striking population differences
with respect to PTB have existed for decades, with women of African
ancestry having a substantially larger burden of risk than women of
European ancestry. The estimated annual cost of PTB in the US alone
was over S26.2 billion in 2005.sup.11. Despite these statistics,
there remains a paucity of effective strategies for predicting and
preventing PTB.
[0005] Although twin studies have documented that maternal and
fetal genetics play roles in determining the length of gestation,
environmental factors, including the microbiome, are the most
important contributors to PTB, particularly among women of African
ancestry.sup.12. Microbe-induced inflammation resulting from
urinary tract infection, sexually transmitted diseases including
trichomoniasis, or bacterial vaginosis is thought to be a cause of
PTB.sup.13-17. Ascension of microbes from the lower reproductive
tract to the placenta, fetal membranes and uterine
cavity.sup.14,18, and hematogenous spread of periodontal pathogens
from the mouth.sup.19-21, have also been invoked to explain the
more than 30% of PTBs that are associated with microbial
etiologies.
[0006] In contrast to other body sites, where high diversity of the
microbiota is generally associated with health, a homogeneous
Lactobacillus-dominated microbiome has long been considered the
hallmark of a healthy female reproductive tract.sup.22. A
microbiome with higher species diversity is considered less
healthy, particularly in women with bacterial vaginosis, which is
characterized by dysbiosis and the presence of variety of bacterial
anaerobes including, but not limited to, Gardnerella vaginalis,
Atopobium vaginae, taxa of the genera Megasphaera, Mobiluncus,
Prevotella, Sneathia, and of the order Clostridiales
(BVAB1/2/3).sup.23-26. However, more advanced technologies have
revealed a more complex picture suggesting that a healthy vaginal
microbiome can sometimes be characterized by a diverse
microbiota.sup.27-35.
[0007] Several studies have examined the relationship of the
vaginal microbiome and the outcome of pregnancy, including but not
limited to PTB.sup.36-46. Collectively, these studies suggest that
the composition of the microbiome of the female reproductive tract
has a significant, population-specific, impact on PTB risk.
However, as of yet, no consistent significant associations have
emerged between specific microbial taxa or combinations thereof and
PTB, as well as other adverse pregnancy outcomes.
SUMMARY
[0008] The present disclosure describes significant harbingers of
adverse pregnancy outcomes such as PTB early in pregnancy or prior
to pregnancy. A high-resolution taxon-specific analyses revealed a
significant association between PTB and the proportional abundance
of TM7-H1, BVAB1 (recently renamed to Lachnovaginosum
genomospecies), Sneathia amnii, and a specific group of Prevotella,
among others. A genomic analysis of these taxa identified virulence
factors, and metatranscriptomic analyses confirmed that genes
encoding these factors are expressed in the vaginal environment.
These taxa were also generally associated with elevated vaginal
levels of inflammatory cytokines, suggesting that complex
host-microbiome interactions likely contribute to PTB. The
disclosure thus provides screening methods having a high degree of
sensitivity and specificity for the prediction of PTB. These
methods allow for the early intervention and prophylaxis of
PTB.
[0009] An aspect of the disclosure provides a method for
determining the risk of an adverse pregnancy outcome such as PTB
for a woman, comprising measuring a level or abundance of TM7-H1
and optionally one or more of BVAB1, Sneathia amnii, and Prevotella
cluster 2 in a vaginal sample obtained from the woman, and
identifying the woman as having an increased risk for PTB if the
levels are increased compared to corresponding standard control
levels. In some embodiments, the level or abundance of each of
TM7-H1, BVAB1, Sneathia amnii, and Prevotella cluster 2 is
measured.
[0010] In some embodiments, the method further comprises measuring
a level or abundance of one or more of Dialister cluster 51
(comprising Dialister OTU30 and Dialister OTU167), Prevotella
amnii, Sneathia sanguinegens, Aerococcus christensenii,
Clostridales BVAB2, Coriobacteriaceae OTU27, Dialister
micraerophilus, Parvimonas OTU142, Megasphaera OTU70 type 1, and
Lactobacillus crispatus cluster (comprising L. crispatus, L.
acidophilus, L. amylovorus, L. gallinarum, L. helveticus, L.
kitasatonis, L. sobrius and L. ultunensis).
[0011] In some embodiments, the vaginal sample is obtained between
6 and 24 weeks of gestation. In other embodiments, the samples are
obtained between 1 and 6 weeks of gestation, or prior to pregnancy
in women who are planning on getting pregnant.
[0012] Another aspect of the disclosure provides a method for
determining the risk of PTB for a woman and administering at least
one PTB prophylactic treatment to a woman identified as being at
risk for PTB, comprising i) measuring a level or abundance of
TM7-H1 and optionally one or more of BVAB1, Sneathia amnii, and
Prevotella cluster 2 in a vaginal sample obtained from the woman;
ii) identifying the woman as having an increased risk for PTB if
the levels or abundances are increased compared to corresponding
standard control levels; and iii) administering the at least one
PTB prophylactic treatment to the woman who is identified as having
an increased risk for PTB.
[0013] In some embodiments, the at least one PTB prophylactic
treatment is selected from the group consisting of antenatal
corticosteroids, antibiotics, tocolytics, progesterone, cerclage
application, feminine hygiene protocols, products that modify the
conditions of the female reproductive tract, prebiotics,
probiotics, microbial or bacteriophage preparations, vaginal
microbial transplants, and combinations thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIGS. 1A-B. Vagitypes of a) 90 women who delivered at term
(.gtoreq.39 wks gestation), and b) 45 women who delivered
prematurely (<37 wks gestation). Profiles were generated as
described in the Example from the earliest sample collected from
each participant. The outer rings identify the 13 community states,
or vagitypes, into which these microbiome profiles fall.
[0015] FIG. 2. Abundance of taxa significantly different in PTB and
TB cohorts from FIG. 1. Box shows median and interquartile range,
whiskers extend from minimum to maximum values within each cohort.
Taxa abbreviations: Lcricl, Lactobacillus crispatus (comprising L.
crispatus, L. acidophilus, L. amylovorus, L. gallinarum, L.
helveticus, L. kitasatonis, L. sobrius and L. ultunensis); BVAB1,
Lachnospiraceae BVAB1; Pcl2, Prevotella cluster 2 (comprising
Prevotella buccalis, Prevotella timonensis, Prevotella OTU46 and
Prevotella OTU47); Samn, Sneathia amnii; Dc151, Dialister cluster
51 (comprising Dialister OTU30 and Dialister OTU167); Pamn,
Prevotella amnii; Ssan, Sneathia sanguinegens; Achr, Aerococcus
christensenii; BVAB2, Clostridiales BVAB2; CO27, Coriobacteriaceae
OTU27; Dmic, Dialister micraerophilus; P142, Parvimonas OTU142.
[0016] FIG. 3. Longitudinal generalized additive mixed effect model
(GAMM) of vaginal microbiome composition during pregnancy. The
model incorporates BMI, ancestry (African or European), pregnancy
outcome (PTB, TB), a smoother for gestational age, and a random
subject effect was used to longitudinally model log-transformed
relative abundances of vaginally relevant taxa. Each panel plots
log-transformed abundances of taxa throughout pregnancy and the
plots are ordered based on the p-values for pregnancy outcome in
the GAMM from highest (top left) to lowest (bottom right).
[0017] FIGS. 4A-B. Sparse Canonical Correlation Analysis (sCCA).
Cytokine abundance in vaginal samples from women who experience TB
(a) or PTB (b) were subjected to an integrative sCCA using
log-transformed cytokine levels and log-transformed taxonomic
profiling data. Triangles represent bacterial taxa and dots
represent cytokines. Note that the component 1 axis for the term
birth sCCA (left) has been reversed for effective visual comparison
with preterm birth sCCA.
[0018] FIGS. 5A-C. Taxa that significantly differ in PTB and TB
cohorts. The distributions of proportional abundance of taxa
significantly differ in PTB (n=31) and TB (n=59) cohorts; the
earliest sample available for each subject within the first 24
weeks of pregnancy was used for each subject. Abundance values
below 0.001 were rounded down to 0. The taxa are: BVAB1:
Lachnospiraceae BVAB1, Pcl2: Prevotella cluster 2, Mty1:
Megasphaera OTU70 type1, Samn: Sneathia amnii, TM7: TM7-H1, Dc151:
Dialister cluster51, Pamn: Prevotella amnii, BVAB2: Clostridiales
BVAB2, Dmic: Dialister micraerophilus and P142: Parvimonas OTU142.
These 10 taxa have p<0.05 to support a significant difference in
proportional abundance between PTB and TB cohorts using a
Mann-Whitney U test (two-sided) and the Benjamini-Hochberg
correction procedure with a False Discovery Rate of 5%. (A) Boxes
show median and interquartile range; whiskers extend from minimum
to maximum values within each cohort. (B, C) Scatter plot of the
PTB predictive score returned by the model (horizontal axis)
plotted against gestational age at birth (vertical axis). Each
point corresponds to a sample from a subject: left bars--PTB
subjects (n=31), right bars--TB subjects (n=59). (C) Shows more
detailed view of the region where majority (48 of 59) of TB samples
are located.
DETAILED DESCRIPTION
[0019] Embodiments of the disclosure provide methods for the
prediction of an elevated risk for an adverse pregnancy outcome
such as preterm birth. The screening tests have high sensitivity
and high specificity, and permit therapeutic intervention to
decrease the risk.
[0020] Adverse pregnancy outcomes include, but are not limited to,
spontaneous miscarriage, spontaneous abortion, preeclampsia, low
birth weight, stillbirth, preterm rupture of membranes (PROM),
preterm premature rupture of membranes (PPROM), and
chorioamnionitis.
[0021] The term "preterm birth" (PTB) may be used interchangeably
with "preterm delivery" (PTD) and refers to childbirth that occurs
before the end of the 37.sup.th week of gestation.
[0022] In one embodiment, the disclosure provides methods for
diagnosing patients at risk for PTB based on the presence or
absence of and/or the relative abundance of particular taxa of
microbes in the vagina. Such patients have a higher than average or
higher than normal chance of delivering a baby before 37 weeks of
gestation as compared to individuals who have different vaginal
microbes, or different amounts of microbes, or different relative
amounts of microbes. Early identification of such a propensity
allows early intervention, e.g. by altering the identity and/or the
relative abundance of vaginal microflora associated with, and
possibly causing, PTB, so that progression to PTB may be avoided,
or delayed, or the associated symptoms may be lessened.
[0023] In some embodiments, a patient being screened by the methods
disclosed herein may be asymptomatic with respect to PTB and may
not have been previously deemed to be susceptible to PTB, e.g.
pregnant nulliparous women or pregnant primiparous or multiparous
women with no history of preterm birth. In other embodiments, a
patient may be asymptomatic with respect to PTB, but for some
reason, may be deemed susceptible to PTB (e.g. due to a previous
history of PTB), and the methods of the invention provide a way to
predict whether or not this is likely to occur. In some
embodiments, the patient may already exhibit overtly one or more
symptoms of PTB, such as a shortening cervix or frequent uterine
contractions. In some embodiments, the patient is not pregnant and
is screened prior to pregnancy.
[0024] In some embodiments, the identification of particular
microflora (e.g. of particular phyla, genera or species of
microbe(s)) may allow targeted therapies directed against the
microbe or microbes which are undesirable, and/or therapies which
increase the amount of desirable microflora, e.g. those which
compete with the undesirable microbes, and/or which supply
activities or produce substances which are beneficial, especially
with respect to PTB.
[0025] In order to practice the methods described herein, generally
a sample of vaginal microflora is obtained from the patient by any
method known to those of skill in the art. The sample may be
obtained from a pregnant woman at any time prior to 37 weeks of
gestation, e.g. prior to 35, 34, 33, 32, 31, 30, 29, 28, 27, 26,
25, or 24 weeks. In some embodiments, the sample is obtained
between 6 and 24 weeks of gestation, inclusive. In some
embodiments, the sample is obtained prior to 6 weeks of gestation.
In other embodiments, the sample is obtained prior to
pregnancy.
[0026] The term "vaginal sample," as used herein, refers to any
vaginal sample suitable for testing or assaying according to the
methods of the present invention. One example of a vaginal sample
can be referred to as a gynecological sample, such as a vaginal
swab obtained according to the procedures accepted in the medical
field. However, the term "vaginal sample" is not limited to vaginal
swabs, but can also be used to describe discharge or mucus samples,
a cervical mucus sample, a cervical swab sample, a tissue sample or
cell samples, obtained, processed, transported and stored using
various suitable procedures. For examples, the samples can be
stored in suitable storage or transportation devices, refrigerated,
frozen, desiccated, diluted, mixed with various additives, or
mounted on slides. In some embodiments, the "sample" may in fact be
a urine sample which in most cases provides an accurate proxy for
the `vaginal sample".
[0027] The sample is tested for the presence or absence of, and/or
for the relative abundance of, at least one bacteria selected from
the group consisting of (with exemplary genomic Genbank accession
numbers provided in parentheses): BVAB1 (also known as
Clostridiales genomosp. BVAB1; PQV000000000), Sneathia amnii
(NZ_CP011280), TM7-H1 (also known as Candidatus Saccharibacteria
genomosp. TM7-H1; CP026537), Prevotella cluster 2 (NZ_ADEF01000048;
Prevotella timonensis-NBAX01000001; Prevotella sp. BV3P1
NZ_AWXC00000000; Prevotella buccalis PNGJ01000001), Dialister
cluster 51 (Veillonellaceae bacterium DNF00626; NZ_KQ960816),
Prevotella amnii (KQ960470), Sneathia sanguinegens (LOQF01000001),
Aerococcus christensenii (CP014159), Clostridales BVAB2 (closest
taxa available Clostridiales bacterium KA00274; KQ959578 [id 95%]),
Coriobacteriaceae OTU27 (Coriobacteriales bacterium DNF00809;
NZ_KQ959671), Dialister micraerophilus (GL878519), Megasphaera
OTU70 type 1 (ADGP01000001.1 and NZ_AFIJ01000040.1), and Parvimonas
OTU142 (closest taxa available Parvimonas sp. KA00067;
NZ_KQ960143.1 [id 88%]) wherein an increase in any of these
bacteria as compared to a control is indicative of a higher risk
for PTB. Prevotella cluster 2 includes at least the following taxa:
Prevotella buccalis, Prevotella timonensis, Prevotella OTU46 and
Prevotella OTU47). In some embodiments, the sample is also tested
for the presence or absence of, and/or for the relative abundance
of Lactobacillus crispatus (comprising L. crispatus, L.
acidophilus, L. amylovorus, L. gallinarum, L. helveticus, L.
kitasatonis, L. sobrius and L. ultunensis) cluster wherein a
decrease in abundance as compared to a control is indicative of a
higher risk for PTB.
[0028] An exemplary gene sequence which encodes for the 16S rRNA of
TM7-H1 is represented by SEQ ID NO: 1. An exemplary gene sequence
which encodes for the 16S rRNA of BVAB1 is represented by SEQ ID
NO: 2. An exemplary gene sequence which encodes for the 16S rRNA of
Sneathia amnii is represented by SEQ ID NO: 3. An exemplary gene
sequence which encodes for the 16S rRNA of Prevotella OTU46 is
represented by SEQ ID NO: 4. An exemplary gene sequence which
encodes for the 16S rRNA of Prevotella buccalis is represented by
SEQ ID NO: 5. An exemplary gene sequence which encodes for the 16S
rRNA of Prevotella timonensis is represented by SEQ ID NO: 6. An
exemplary gene sequence which encodes for the 16S rRNA of
Prevotella OTU47 is represented by SEQ ID NO: 7. An exemplary gene
sequence which encodes for the 16S rRNA of Dialister OTU30 is
represented by SEQ ID NO: 8. An exemplary gene sequence which
encodes for the 16S rRNA of Dialister OTU167 is represented by SEQ
ID NO: 9. An exemplary gene sequence which encodes for the 16S rRNA
of Prevotella amnii is represented by SEQ ID NO: 10. An exemplary
gene sequence which encodes for the 16S rRNA of Sneathia
sanguinegens is represented by SEQ ID NO: 11. An exemplary gene
sequence which encodes for the 16S rRNA of Aerococcus christensenii
is represented by SEQ ID NO: 12. An exemplary gene sequence which
encodes for the 16S rRNA of Clostridales BVAB2 is represented by
SEQ ID NO: 13. An exemplary gene sequence which encodes for the 16S
rRNA of Coriobacteriaceae OTU27 is represented by SEQ ID NO: 14. An
exemplary gene sequence which encodes for the 16S rRNA of Dialister
micraerophilus is represented by SEQ ID NO: 15. An exemplary gene
sequence which encodes for the 16S rRNA of Megasphaera OTU70 type 1
is represented by SEQ ID NO: 16. An exemplary gene sequence which
encodes for the 16S rRNA of Parvimonas OTU142 is represented by SEQ
ID NO: 17. An exemplary gene sequence which encodes for the 16S
rRNA of Lactobacillus crispatus strain NCTC4 is represented by SEQ
ID NO: 18. An exemplary gene sequence which encodes for the 16S
rRNA of Lactobacillus crispatus strain ST1 is represented by SEQ ID
NO: 19. An exemplary gene sequence which encodes for the 16S rRNA
of Lactobacillus acidophilus is represented by SEQ ID NO: 20. An
exemplary gene sequence which encodes for the 16S rRNA of
Lactobacillus amylovorus is represented by SEQ ID NO: 21. An
exemplary gene sequence which encodes for the 16S rRNA of
Lactobacillus gallinarum is represented by SEQ ID NO: 22. An
exemplary gene sequence which encodes for the 16S rRNA of
Lactobacillus helveticus is represented by SEQ ID NO: 23. An
exemplary gene sequence which encodes for the 16S rRNA of
Lactobacillus kitasatonis is represented by SEQ ID NO: 24. An
exemplary gene sequence which encodes for the 16S rRNA of
Lactobacillus sobrius is represented by SEQ ID NO: 25. An exemplary
gene sequence which encodes for the 16S rRNA of Lactobacillus
ultunensis is represented by SEQ ID NO: 26.
[0029] In some embodiments, the level of at least one bacteria in
the sample is measured. In some embodiments, a plurality, i.e. 2 or
more, is measured, e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14 or more. It is contemplated that the levels of bacteria
other than those described herein may be measured in addition to
those described herein. In some embodiments, the relative amount of
bacteria in the sample is measured and compared to the relative
amount in a control sample.
[0030] As used herein, "increase" or "decrease" refers to
increasing or lowering by, for example, at least about 5%, 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more when compared to at
least one type of standard or control.
[0031] Diagnostic methods or tests according to some embodiments
use bacterial markers that have high specificity and/or high
sensitivity. The terms "sensitivity" and "specificity" are used
herein to refer to statistical measures of the performance of
diagnostics tests. Sensitivity refers to a proportion of positive
results which are correctly identified by a test. Specificity
measures a proportion of the negative results that are correctly
identified by a test. The term "high specificity" refers to
specificity that is equal to or over 70%, 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The term
"high sensitivity" refers to sensitivity that is equal to or over
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99%.
[0032] Negative control samples for use in establishing negative
standards of women unlikely to experience PTB (e.g. a "normal" or
"healthy" vaginal microbiome signature) may be obtained from one or
more subjects not at risk for PTB, for example, pregnant women who
have exceeded a gestational age of 37 weeks, women who have
delivered a baby at full term (i.e., at greater than 37 weeks), or
non-pregnant women. Alternatively, or in addition, positive control
samples may be used, e.g. samples obtained from subjects who have
experienced PTB and/or are know to be at risk of PTB, to establish
positive standards for women likely to experience PTB (e.g. an "at
risk" vaginal microbiome signature). Further, standards may be
refined to include specific stratified categories, examples of
which include "very high risk" (PTB will occur without
intervention), "moderate risk" (PTB is likely without
intervention), and "low risk" (PTB is possible and the patient
should be monitored but does not necessarily need intervention at
the time). The risk levels may also take into account one or more
factors in addition to the microbial signature, such as age, ethnic
background, socioeconomic level, overall health, previous health
records (including prior births with or without occurrences of
PTB), etc.
[0033] Detection of microbes may be done in any of a number of ways
that are known to those of ordinary skill in the art, including but
not limited to culturing the organism(s), conducting various
analyses which are indicative of the presence of the microbe(s) of
interest (e.g. by microscopy, using staining techniques, enzyme
assays, antibody assays, etc.), or by sequencing of genetic
material (DNA or RNA) using, e.g. NextGen or Xgen technology, qPCR,
chip technology, and others. While any category (or categories) of
nucleic acid(s) may be detected (usually amplified using, e.g. PCR
techniques), particularly useful amplification strategies include
the use of primers (e.g. universal primers) which amplify ribosomal
RNA genes (rRNA) as is known in the art. Other useful technologies
include metagenomic or metatranscriptomic sequencing, in which all
the DNA or RNA, respectively, in a sample is sequenced and used for
taxonomic classification and determinations of abundance or
relative abundance.
[0034] It will be appreciated that determining the abundance of
microbes may be affected by taking into account any feature of the
microbiome. Thus, the abundance of microbes may be affected by
taking into account the abundance at different phylogenetic levels;
at the level of gene abundance; gene metabolic pathway abundances;
sub-species strain identification; SNPs and insertions and
deletions in specific bacterial regions; growth rates of bacteria,
the diversity of the microbes of the microbiome, etc.
[0035] In some embodiments, determining a level or set of levels of
one or more types of microbes or components or products thereof
comprises determining a level, abundance or relative abundance, or
set of levels, abundances, or relative abundances of one or more
DNA or RNA sequences. In some embodiments, one or more DNA or RNA
sequences comprises any DNA or RNA sequence that can be used to
differentiate between different microbial types. In certain
embodiments, one or more DNA or RNA sequences comprises 16S rRNA
gene or 16S rRNA sequences. In certain embodiments, one or more DNA
or RNA sequences comprises 18S rRNA gene or 18S rRNA sequences. In
some embodiments, 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 100, 1,000,
5,000 or more sequences are amplified. In some embodiments one or
more DNA or RNA sequences comprises metagenomic or
metatranscriptomic sequences of all the DNA or RNA in a sample.
[0036] 16S and 18S rRNA gene sequences encode small subunit
components of prokaryotic and eukaryotic ribosomes respectively.
rRNA genes are particularly useful in distinguishing between types
of microbes because, although sequences of these genes differs
between microbial species, the genes have highly conserved regions
for primer binding. This specificity between conserved primer
binding regions allows the rRNA genes of many different types of
microbes to be amplified with a single set of primers and then to
be distinguished by amplified sequences.
[0037] In some embodiments, in order to classify a microbe as
belonging to a particular genus, family, order, class or phylum, it
must comprise at least 70% sequence homology, at least 75% sequence
homology, at least 80% sequence homology, at least 85% sequence
homology, at least 90% sequence homology, at least 91% sequence
homology, at least 92% sequence homology, at least 93% sequence
homology, at least 94% sequence homology, at least 95% sequence
homology, at least 96% sequence homology, at least 97% sequence
homology, at least 98% sequence homology, at least 99% sequence
homology to a reference microbe known to belong to a particular
taxon.
[0038] In some embodiments, in order to classify a microbe as
belonging to a particular species, it must comprise at least 90%
sequence homology, at least 91% sequence homology, at least 92%
sequence homology, at least 93% sequence homology, at least 94%
sequence homology, at least 95% sequence homology, at least 96%
sequence homology, at least 97% sequence homology, at least 98%
sequence homology, at least 99% sequence homology to a reference
microbe known to belong to the particular species.
[0039] Once a patient is identified being at an elevated risk of
PTB, suitable clinical intervention can be undertaken to
prophylactically treat PTB. Exemplary treatments include but are
not limited to: eliminating or lessening microflora which are
increased in patients with PTB (e.g. using antibiotics or other
therapies), promoting or increasing microflora which are decreased
in patients with PTB (e.g., using prebiotics, probiotics, or other
therapies), antenatal corticosteroids, tocolytics, progesterone,
cerclage application, products that modify the conditions of the
female reproductive tract (e.g., soaps such as those produced by
Summer's Eve.RTM.), genetically engineered microbial or
bacteriophage preparations, vaginal microbial transplants (e.g.,
with one or more species known to be associated with or conducive
to a healthy microbiome that is not associated with PTB), and
combinations thereof. Examples of microbes suitable for vaginal
microbial transplants include but are not limited to: Lactobacillus
species, including but not limited to L. crispatus, L. jensenii, L.
gasseri, L. acidophilus, Lactobacillus GG, Lactobacillus rhamnosus,
other Lactobacillus taxa, Bifidobacterium bifidum or other
Bifidobacterium taxa, or other bacterial taxa.
[0040] A pregnant patient or subject to be treated by any of the
methods of the present disclosure can mean either a human or a
non-human animal including, but not limited to dogs, horses, cats,
rabbits, gerbils, hamsters, rodents, birds, aquatic mammals,
cattle, pigs, camelids, and other zoological animals.
[0041] In some embodiments, the treatment is administered to the
subject in a therapeutically effective amount. By a
"therapeutically effective amount" is meant a sufficient amount of
active agent to decrease the likelihood of PTB at a reasonable
benefit/risk ratio applicable to any medical treatment. In some
aspects, PTB is prevented. In other aspects, the gestational time
is increased until the infant has a reasonable chance of survival
outside the uterus, i.e. at least up to about 22 weeks, and
preferably longer, such as up to about 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 weeks.
[0042] Embodiments of the disclosure also provide methods for
monitoring the efficacy of a treatment protocol that is ostensibly
treating PTB. This might be early in pregnancy, or in some cases in
women who are anticipating or planning on getting pregnant. The
method involves determining vaginal microbiome signatures of a
patient who is or who is going to be pregnant, and who may be
treated to prevent PTB. Multiple signatures are generally obtained
and analyzed at suitable time intervals, e.g., just prior to
treatment to establish a baseline, and then repeatedly every few
days or weeks thereafter. Subsequent signatures are compared to
suitable reference signatures and/or to one or more previous
signatures from the patient. If subsequent signatures indicate that
the patient's vaginal microfloral signature is improving (e.g., is
more similar to that of control subjects as described herein) then
the treatment may be continued without adjustment, or may be
gradually decreased, and may even be discontinued. However, if no
improvement is observed, or if a signature indicates a worsening of
the condition, then the treatment protocol can be adjusted
accordingly, e.g., more of a treatment agent may be administered,
or a different and/or more drastic form of treatment may be
implemented, etc. The microflora signature is thus used to assess
treatment adequacy and treatment response.
[0043] Embodiments of the present disclosure also include kits for
use in the screening of PTB risk. For instance, such kits may
include primer sets for the detection, amplification and
classification of the bacterial strains present in a test sample
taken from a host. The primer sets in such kits may include
taxon-specific primer sets for classification of the bacteria
present in the test sample. For instance, a kit may include primers
that would allow for identification and classification of bacterial
strains as described herein that are present in the test sample, so
that comparison of relative amounts of bacteria present in the test
sample can be determined and compared to a predetermined standard.
Alternative kits may have home versions wherein antibodies,
specific oligonucleotides, or other molecular methods for specific
identification of a bacterial species, strain or taxon. These could
be available as disposable dipsticks, akin to home pregnancy tests.
Vaginal swabs, urine samples, or other appropriate specimens can be
exposed to such dipsticks to detect and quantify bacterial taxa in
the sample.
[0044] Before exemplary embodiments of the present invention are
described in greater detail, it is to be understood that this
invention is not limited to particular embodiments described, and
as such may, of course, vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to be limiting, since the
scope of the present invention will be limited only by the appended
claims.
[0045] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0046] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, representative illustrative methods and materials are
now described.
[0047] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference and are incorporated herein by reference
to disclose and describe the methods and/or materials in connection
with which the publications are cited. The citation of any
publication is for its disclosure prior to the filing date and
should not be construed as an admission that the present invention
is not entitled to antedate such publication by virtue of prior
invention. Further, the dates of publication provided may be
different from the actual publication dates which may need to be
independently confirmed.
[0048] It is noted that, as used herein and in the appended claims,
the singular forms "a", "an", and "the" include plural referents
unless the context clearly dictates otherwise. It is further noted
that the claims may be drafted to exclude any optional element. As
such, this statement is intended to serve as antecedent basis for
use of such exclusive terminology as "solely," "only" and the like
in connection with the recitation of claim elements, or use of a
"negative" limitation.
[0049] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present invention. Any recited
method can be carried out in the order of events recited or in any
other order which is logically possible.
[0050] The invention is further described by the following
non-limiting examples which further illustrate the invention, and
are not intended, nor should they be interpreted to, limit the
scope of the invention.
Example
Summary
[0051] Reported herein is an analysis of data generated in a
collaborative effort under the umbrella of the National Institutes
of Health's integrative Human Microbiome Project iHMP.sup.47,
representing a large cohort of pregnant women with deep
characterization of the vaginal microbiome and risk of PTB. We
identified differences in the vaginal microbial communities of
women who experienced PTB, identified bacterial taxa associated
with these differences, and showed that these bacteria express
genes with pathogenic potential, supporting the concept that they
elicit early induction of labor directly by infection, or
indirectly by inducing inflammatory responses.
Methods
Participant Enrollment, Informed Consent, and Health History
Collection
[0052] Participants for this study were enrolled from women
visiting maternity clinics in Virginia and Seattle. All study
procedures involving human subjects were reviewed and approved by
the institutional review board at Virginia Commonwealth University
(IRB #HM15527). Participants, approximately 1500, were enrolled
from Women's Clinics at VCU Health Center, approximately 1000
women, and at multiple sites in Washington state, approximately 500
women, state by our partner registry, the Global Alliance to
Prevent Prematurity and Stillbirth (GAPPS). Study protocols were
harmonized across sites, and data and samples were all distributed
to the VCU study for analysis. All study participants enrolled in
Virginia and Washington were also enrolled in the Research Alliance
for Microbiome Science (RAMS) Registry at Virginia Commonwealth
University. RAMS Registry protocols were approved at Virginia
Commonwealth University (IRB #HM15528); GAPPS-associated sites
ceded review to the VCU IRB through reliance agreements. The study
was performed with compliance to all relevant ethical regulations.
Written informed consent was obtained from all participants and
parental permission was obtained for participating minors.
[0053] Pregnant women and minors at least 15 years of age were
provided literature on the project and invited to participate in
the study. At initial visits, women over 18 or minors between 15
and 18 were administered informed consent or informed assent and
parental permission, respectively. Women/minors who: 1) were
incapable of understanding the informed consent or assent forms, or
2) were incarcerated were excluded from the study. Comprehensive
demographic, health history and dietary assessment surveys were
administered, and relevant clinical data (gestational age, height,
weight, blood pressure, vaginal pH, diagnosis, etc.) was recorded.
Relevant clinical information, e.g., gestational age, weight, any
diagnosis, etc., was also obtained from neonates at birth and
discharge.
[0054] At subsequent prenatal visits, triage, in labor and
delivery, and at discharge, additional surveys were administered,
relevant clinical data was recorded, and samples were collected.
Samples included mid-vaginal, buccal, rectal, and skin swabs,
urine, blood, cord, cord blood, placenta, membranes and amniotic
fluid, baby buccal, baby rectal, baby skin, baby meconium and first
stool. Vaginal and rectal samples were not collected at labor and
delivery or at discharge. Women with any of the following
conditions were excluded from sampling at a given visit:
1. Incapable of self-sampling due to mental, emotional or physical
limitations. 2. More than minimal vaginal bleeding as judged by the
clinician. 3. Ruptured membranes prior to 37 weeks. 4. Active
herpes lesions in the vulvovaginal region.
Case/Control Design
[0055] We selected 47 preterm cases of singleton, non-medically
indicated preterm births from women who delivered between 20 and
<37 weeks gestation from the women who enrolled in the Virginia
arm of the study. The participants had completed the study through
delivery, and their gestational age information had been recorded
in the study operational database as of July 2016. We case-matched
the preterm participants 2:1 with participants who completed the
study with singleton term deliveries .gtoreq.39 weeks matching
based on ethnicity, age and income. We matched cases as close to
exact matches as possible. Most cases were matched using the
in-house script; a few difficult-to-match cases were matched by
hand. Case matching was performed blinded to all other study data.
Two of the 47 preterm births did not have 16S rRNA that passes QC,
thus these PTB samples and their controls were excluded from the
taxonomic 16S rRNA analyses (FIG. 1) and demographic data in Table
1.
Sample Collection
[0056] Samples (i.e., buccal, mid-vaginal wall, cervical, and
rectal) were collected from appropriately consented women at the
enrollment visit, longitudinally at each prenatal visit, at triage,
and at labor and delivery. Samples were collected with BD BBL.TM.
CultureSwab.TM. EZ swabs. Vaginal and rectal swabs were collected
either by a clinician during a pelvic exam or by self-sampling.
Cervical samples, when collected, were collected by a clinician
during a pelvic exam using a speculum for the vaginal samples.
Research coordinators collected buccal samples, and instructed the
participants on self-sampling procedures, provided a self-sampling
instructional brochure, and provided the participant a room for
self-sampling. When samples were self-collected, no cervical
samples were obtained. Self-sampling has been shown to provide
samples equivalent to those collected by a trained
clinician.sup.58.
[0057] Samples were collected as follows: 1) mid-vaginal wall: a
double-tipped CultureSwab.TM.EZ swab was placed carefully on the
vaginal sidewall about halfway between the introitus and the
cervix, pressed firmly into the sidewall to a depth of roughly the
diameter of the swab, and rolled dorsally-ventrally back and forth
four times to coat the swab, and removed; 2) cervical: a
single-tipped CultureSwab.TM.EZ swab was inserted into the
endocervix to the depth of the entire tip of the swab, rotated 360
degrees, held for ten seconds, and removed, being careful not to
contact the vaginal walls; 3) buccal: a double-tipped
CultureSwab.TM.EZ was placed firmly in the mid-portion of the cheek
and rubbed up and down 10 times while constantly rotating, and
removed; 4) rectal: a CultureSwab.TM.EZ swab was inserted .about.1
inch into rectum, rotated 360 degrees, held for ten seconds, and
removed. Vaginal pH was collected using commercial applicators with
pH paper. Briefly, the sterile applicators, miniature applicators
with pH indicator affixed to the tip, were inserted .about.1.5-2
inches into vagina, applied gently to vaginal wall, and withdrawn.
The pH was read by comparing the color of the pH indicator to a
color chart by the Research Coordinator.
[0058] Samples from neonates (buccal, rectal, meconium and first
stool) were collected at birth, at discharge, and in the NICU (if
admitted). Buccal swabs were collected from neonates essentially as
described above. Rectal swabs were collected using single-tip
CultureSwab.TM.EZ swab inserted .about.1/4 inch into the rectum as
described above. Meconium and first stool was collected from
diapers using a sterile swab to collect a few grams of
material.
Sample Processing
[0059] After collection, swabs were immediately immersed in
transfer buffer depending on the objectives; i.e., swabs for DNA
isolation were immersed in MoBio PowerSoil.RTM. DNA Isolation
buffer, swabs for RNA purification were immersed in RNAlater.RTM.
(Qiagen), and swabs for cytokine profiling were immersed in 10 mM
Tris (pH 7.0), 1 mM EDTA. Swabs were either processed immediately
or stored until processing at -80.degree. C. Swabs for DNA
purification were processed using the MoBio PowerSoil.RTM. Kit,
essentially as described by the manufacturer. Swabs for RNA
purification were processed using the MoBio PowerMicrobiome.TM. RNA
Isolation Kit as described by the manufacturer. Total RNA was
depleted of human and microbial rRNA using the Epicentre/Illumina
Ribo-Zero.TM. Magnetic Gold Kit as described by the manufacturer.
DNA and RNA samples were stored at -80.degree. C.
16S rRNA Taxonomic Surveys of the Vaginal Microbiome
[0060] Each DNA sample was amplified with barcoded primers
validated for vaginal taxa as previously reported.sup.48. Samples
were multiplexed (384 samples/run) and sequenced using 2.times.300
b PE technology on an Illumina MiSeq.RTM. sequencer to generate a
depth of coverage of at least 50,000 reads per sample. The raw
sequence data was demultiplexed into sample paired-end fastq files
based on unique barcode sequences using custom python script. The
preprocessing of sequences was performed using the MeFiT.sup.59
pipeline, with amplicons (on average .about.540 bp long) generated
by merging the overlapping tails of paired-end sequences followed
by quality filtering using a meep (maximum expected error rate)
cutoff of 1.0. Non-overlapping high-quality reads were screened for
chimeric sequences with UCHIME.sup.60 against our custom database
of vaginally relavant taxa. Each processed 16S rRNA gene sequence
was taxonomically classified to the species-level by using
STIRRUPS.sup.48 by aligning against a custom reference database
using USEARCH.sup.61. Reference sequences for Prevotella cluster 2
include Prevotella buccalis, Prevotella timonensis, Prevotella
OTU46 and Prevotella OTU47. Only samples with at least 1,000 reads
that met filtering criteria were analyzed.
Custom 16S rRNA V1-V3 Reference Database for Vaginal/Rectal
Comparison
[0061] Full-length 16S rRNA gene sequences were collected from
various sources--(i) the All-species Living Tree Project (Silva:
LTPs123_SSU).sup.62, (ii) STIRRUPS.sup.48 database, and (iii) Human
Oral Microbiome Database (HOMD).sup.63. Since the partial 16S rRNA
V1-V3 region is not distinct enough to get species-level
stratification for certain bacterial genera, we extracted the V1-V3
region from every reference sequence using V-Xtractor.sup.64, a
tool that identifies the hypervariable regions using Hidden Markov
Models (HMMs). These partial sequences were then clustered into
representative sequence set at 99% identity using UCLUST.sup.61.
The representative sequences were annotated to the least common
ancestor (LCA) taxonomic-level, in cases where the cluster
comprised sequences from different bacterial species. The dataset
was then supplemented by partial V1-V3 sequences from the STIRRUPs
database, especially ones for which full length 16S rRNA sequence
is not available. This resulted in a set of 9,299 representative
16S rRNA V1-V3 partial sequences. First sample visits (FIG. 1) were
used to compare the proportional abundance of taxa in vaginal and
rectal samples. Samples for 11 subjects did not have 16S rRNA
profiles that passed the QC, thus 124 samples were used for this
comparative analysis.
Whole Metagenomic/Metatranscriptomic Sequencing
[0062] DNA libraries were prepared using KAPA Biosystems
HyperPlus.RTM. Library Kit and sequenced on an Illumina HiSeq.RTM.
4000 (2.times.150 b PE). We multiplexed 24 samples per lane and
obtain .about.1-2.times.10.sup.7 150 nt reads per sample. The rRNA
depleted messenger RNA was prepared for sequencing by constructing
cDNA libraries using the KAPA Biosystems KAPA RNA HyperPrep.RTM.
Kit. Indexed cDNA libraries were pooled in equimolar amounts and
sequenced on the Illumina HiSeq.RTM. 4000 instrument running 4
multiplexed samples per lane with an average yield of .about.100
Gb/lane, sufficient to provide >100.times. coverage of the
expression profiles of the most abundant 15-20 taxa in a
sample.
Whole Shotgun Metagenomic/Metatranscriptomic Data
Pre-Processing
[0063] Raw sequence data was demultiplexed into sample-specific
fastq files using bcl2fastq conversion software from Illumina.
Adapter residues were trimmed from both 5' and 3' end of the reads
using Adapter Removal tool v2.1.3.sup.65. The sequences were
trimmed for quality using meeptools.sup.66, retaining reads with
minimum read length of 70b and meep (maximum expected error)
quality score less than 1. Human reads were identified and removed
from each sample by aligning the reads to hg19 build of the human
genome using the BWA aligner.sup.67.
Functional Analysis of Metagenomic and Metatranscriptomic Sequence
Reads
[0064] Assignment of metagenomic and metatranscriptomic sequence
reads to known genes/pathways was performed using ASGARD.sup.68,
HUMAnN2.sup.69 and ShortBRED.sup.70. These reads are also compared
to appropriate databases (KEGG, GO, COG, etc.) using BLAST.sup.71
or other alignment tools to characterize functional data about
these samples.
BVAB1 Genome Assembly from Metagenomic Reads
[0065] Starting with high quality trimmed metagenomic sequence
reads from one sample with a high abundance of BVAB1, human reads
were removed by alignment to the human hg19 reference sequence
using BWA.sup.67 alignment software. The remaining reads were
processed as described below. The reads were digitally normalized
with BBMap (sourceforge.net/projects/bbmap/) with a target coverage
of >40.times. coverage to remove reads from highly repetitive
elements of the genomes that may hamper the de novo assembly
process and to ensure that reads originating from PCR duplication
are excluded prior to assembly. Reads were assembled with SPAdes
ver 3.8.0.sup.72,73 using the "-meta" option to generate a
consensus assembly scaffold. Prior to clustering the scaffolds
generated by SPAdes version 3.8.0, the human depleted reads were
aligned back to the scaffolds using Bowtie2.sup.74 with the
`-very-sensitive` option for global alignment. The resulting bam
files were converted into "scaffold-to-average coverage" maps using
a custom Python script. These contigs were clustered into
individual genomes using MyCC.sup.75 with tetramer frequencies
coupled with the average coverage. Assemblies were identified by
alignment with cpn60 or grpE genes. Note that ribosomal genes are
too similar to segregate into different clusters. Reads were mapped
back to individual MyCC clusters and then submitted to a new
assembly using Newbler Assembler v2.8. Where necessary, gaps were
closed by sequencing of PCR amplicons using primers directed to
contig ends. Prokka.sup.76 and ASGARD.sup.68 were run on each
assembly to find its gene repertoire and annotate it.
TM7-H1 Genome Assembly from Metagenomic Reads Using PacBio Single
Molecule Long Read Technology.
[0066] DNA from a sample with high proportional abundance of TM7
was sent to Jonas Korlach (at Pacific Biosciences) for PacBio
sequencing using the TdT protocol, which is suitable for sequencing
of low-input samples. An HGAP metagenome assembly was performed
using a white list to exclude reads mapped to human yielded three
TM7 contigs. PCR amplication was performed across contigs gaps.
Identification of Virulence and Defense Genes of BVAB1, TM7-H1,
Prevotella timonensis and Sneathia amnii
[0067] The genome sequences were annotated using an in-house
pipeline that utilizes existing tools (e.g., Prokka.sup.76,
ASGARD.sup.68, tRNAScan.sup.77, RNAmmer.sup.78). We also submitted
sequences to the Rapid Annotation using Subsystem Technology (RAST)
server.sup.79 for genome annotation, which classified annotated
genes into broad functional subsystems. We manually curated a
virulence and defense functional supersystem category which
included categories of genes that may cause pathogenic outcome,
persistence, and/or help defend organisms from host and other
bacterial species attack mechanisms. RAST identified functional
subsystem categories in this curated supersystem included:
virulence disease and defense, iron acquisition and metabolism,
motility and chemotaxis, dormancy and sporulation, stress response,
and sulfur metabolism. The list of genes in the supersystem were
then used to compare across genomes and further used in comparisons
of transcription levels of virulence genes in vaginal microbial
profiles that included these taxa.
Transcript Abundances of BVAB1, TM7-H1, Prevotella timonensis, and
Sneathia Amnii
[0068] Transcriptomic profiling of BVAB1, TM7-H1 Prevotella
timonensis, Sneathia amnii was performed using PanPhlAn.sup.80, a
pangenome-based tool. First, we generated species-specific
pangenomes; metagenomic reads were mapped against the corresponding
pangenome using the software to obtain a gene family
presence/absence matrix specific to a strain in a sample based on
the coverage of all genes in a gene family cluster. For the samples
for which we had matching metagenomics and metatranscriptomics data
of the species at a minimum of 2.times. coverage, strain-specific
transcriptional rates based on the gene family profiles from
metagenomic sample data were generated using PanPhlAn. Virulence
gene families were annotated by mapping to the virulence and
defense functional supersystem category as called by RAST genome
annotation of reference genomes.
Metabolic Modeling
[0069] Draft constraint-based metabolic models for TM7, BVAB1, and
Lactobacillus crispatus were generated using functional annotation
information using EC numbers to describe function and KEGG IDs for
nomenclature.
Cytokine Profiling
[0070] The Bio-Plex Pro Human Cytokine 27-plex Assay panel
(M50-0KCAF0Y, Bio-Rad, Hercules, Calif.) was used to measure
cytokine concentrations according to manufacturer's protocol.
Briefly the frozen vaginal swab samples were thawed on ice and
centrifuged at 10,000.times.g for 10 min at 4.degree. C. and
diluted 4 fold in 100 mM Tris buffer, pH 7.5. The assay was carried
out on a black 96-well plate (10021013, Bio-Rad, Hercules, Calif.),
and 50 .mu.l of cytokine standard, inter assay QC control
(described below) and sample was added in duplicate to appropriate
wells. The Bio-Plex MAGPIX.RTM. Multiplex Reader was used for data
acquisition with default settings. Bio-Plex Manager 6.0 software
was used for data analysis using five parameter logistic (5-PL)
non-linear regression model upon optimization for all analytes
within 70-130% recovery range.
[0071] The inter assay QC control was prepared from LPS stimulated
cell culture medium. Briefly VK2/E6E7 (ATCC CRL-2616, ATCC
Manassas, Va.) cells were initially grown in T75 flasks in DMEM/F12
supplemented with 10% FBS (11320-033, 26140079, ThermoFisher,
Waltham, Mass.) at 37.degree. C., 5% CO2 to confluency. These cells
were trypsinized and reseeded at concentration of 3.times.10.sup.5
cells/ml per well on a 24-well plate (82050-892, VWR Radnor, Pa.).
After 24 hrs, the medium was replaced with the fresh medium
containing 100 ng/ml LPS (L2630-10MG, Sigma, St. Louis, Mo.).
Twenty four hours post LPS treatment the cell culture medium was
harvested, pooled and centrifuged at 3,000 rpm.times.10 min at
4.degree. C. The resultant soluble fraction was aliquoted and
stored at -80.degree. C. for use as assay QC.
[0072] Out-of-range cytokine concentration values were imputed with
the upper or lower limit of detection for the specific cytokine
where necessary. Nine cytokines--IL-1b, Eotaxin, IL-8, TNF-.alpha.,
IL-17A, MIP-1b, IL-6, IP-10, RANTES--had fewer than 30%
out-of-range values and were selected for analysis.
Statistical Analyses
Community State Types (CSTs)/Vagitypes
[0073] Vaginal 16S rRNA profiles were assigned to CST/vagitype
based on the taxon with the largest proportion of reads. Samples
where the largest proportion was less than 30% were not assigned a
CST/vagitpe. This "predominant taxon" rule has been shown to
exhibit over 90% agreement with clustering-based methods across a
variety of vaginal microbiome datasets.sup.81, yet is not
population or dataset dependent and is therefore more conducive to
use in a clinical setting. Differences in the numbers of L.
crispatus CSTs among the PTB and TB cohorts was tested with a
Fisher's exact test.
Markov Chain Analysis
[0074] The R package msm was used to fit a continuous-time Markov
chain model for CST transitions. The model takes as input the
subject, CST/vagitype, and gestational age in days for each sample.
The states were L. crispatus, L. iners, BVAB1, G. vaginalis, and
"Other". The pregnancy outcome (PTB, TB) was included as a
co-variate. Because of low numbers of observed transitions between
certain states, only point estimates (and not confidence intervals)
were derived.
Filtering Out Low-Abundant Taxa
[0075] As the first step in analyzing each dataset of vaginal 16S
rRNA profiles, we analyzed the abundance of each taxa present in
the profiles, and removed from further consideration low abundant
species. We used two abundance criteria: we retained taxa that
either a) 5% of the profiles exhibited an abundance of at least 1%,
orb) at least 15% of profiles exhibited an abundance of at least
0.1%. Taxa that failed to meet both a) and b) were removed.
Univariate Analysis to Identify Taxa Significantly Different in
Abundance in Preterm and Term Birth Cohorts
[0076] We analyzed vaginal 16S rRNA profiles from 135 participants,
45 who delivered preterm and 90 who delivered term. The microbiome
profile of the earliest sample from each of these women was used in
this analysis. In this data set, 26 taxa remained after filtering
out of the low-abundance taxa. For each of these 26 most abundant
taxa, we performed a Mann-Whitney U test to identify significant
differences in presence and abundance in preterm and term birth
cohorts. For this analysis, abundance values below 0.00001 were
rounded to zero. Taxa abundance was considered significantly
different between cohorts if the q-value was less than a false
discovery rate (FDR) of 5% after correction via the
Benjamini-Hochberg procedure.sup.82. For each taxon, we also
calculated median and 75-percentile in the preterm and term birth
cohorts.
Longitudinal Models
[0077] A generalized additive mixed model (GAMM).sup.83
incorporating BMI, ethnicity (African, European), pregnancy outcome
(pre-term, full-term), a smoother for gestational age, and a random
subject effect was used to longitudinally model log transformed
relative abundances of vaginally relevant taxa (FIG. 3). Effect
contributions were determined using ANOVA tests. The degree of
smoothness for gestational age was estimated by restricted maximum
likelihood.sup.84. Models were fit using the gamm4: Generalized
additive mixed models using mgcv and lme4. R package package in R
released by Wood and Scheipl in 2017.
Canonical Correlation Analysis of Cytokines and Vaginal Microbiome
Profiles
[0078] An integrative analysis of both log-transformed 16S rRNA
survey data and log-transformed cytokine data was performed using
sparse canonical correlation analysis.sup.85,86 (sCCA, FIG. 4).
Classical canonical correlation analysis.sup.87 explores the
correlation between two sets of quantitative variables measured on
the same subjects. sCCA introduces an 11 penalization term to
handle the case of more variables than observations. Nine
cytokines, with fewer than 30% out-of-range values, were selected
for analysis (IL-1b, Eotaxin, IL-8, TNF-.alpha., IL-17A, MIP-1b,
IL-6, IP-10, RANTES). Out-of-range cytokine concentration values
were then imputed with the upper or lower limit of detection for
the specific cytokine where appropriate. For each subject, the
observation corresponding to the earliest gestational age per
trimester was used for analysis. We perform sCCA separately for
full term and pre-term subjects using the sgcca function in the R
package mixOmics.sup.88.
[0079] The results of sCCA are displayed in a correlation circle
plot.sup.87. The coordinates of the plotted points (variables) are
the correlations between the variables and their canonical
variates. Variables that are strongly positively correlated are
projected close to each other on the plot while variables that are
negatively correlated are plotted opposite each other. The greater
the distance from the origin, the stronger the relationship among
variables.sup.87. The correlation circle plots are constructed
using the plotVar function in the R package mixOmics.
Taxa Co-Occurrence
[0080] Bacterial taxa were determined to be present if they
comprised greater than or equal to 0.1% of the total vaginal
microbiome profile. We utilized the statistical tool REBACCA.sup.89
to mitigate the effects of relative constraint. REBACCA was run
using 50 bootstraps and a visualization of bacterial correlations
was generated using Gephi.sup.90. Correlations with greater than
0.30 or less than -0.3 are shown with negative correlations. Edge
weights are representative of the strength of correlation between
taxa and the four major predictive taxa.
Predictive Modeling of Preterm Birth Using Early-Pregnancy
Microbiome Profiles
[0081] We constructed a linear predictive model of preterm birth as
follows. From the full cohort, we selected subjects who had at
least one vaginal 16S rRNA sample early in the pregnancy, in the
day 42-day 167 (inclusive) gestational age range. A total of 31 PTB
and 59 TB subjects had at least one sample in this time window; if
multiple samples were present in that window, we used the earliest
sample.
[0082] We first filtered out low-abundant species in this dataset:
25 passed the selection criteria. For these taxa, the abundance
data was soft-thresholded with 0.001 threshold, to reduce the
impact of statistical noise resulting from low-abundance values, by
subtracting 0.001 from the abundance and setting all resulting
negative values to 0, and log-transformed through a transform log
10((abundance+0.001)/0.001), where dividing by 0.001 shifts the
logarithm values for abundances in the zero (0.0) to one (1.0)
range from negative to non-negative values. Ten taxa were
significantly different between the PTB and TB cohorts.
[0083] The model construction uses a two-step procedure. First, we
applied a Mann-Whitney U test to all species that survived the
abundance-based filtering criteria, retaining species with a
two-sided p-value of 0.05 or less. Based on these species, the
predictive model was trained using logistic regression with L.sub.1
regularization.sup.91, to reduce the impact of collinearity between
species and the resulting sign-reversals and false detections.
Regularized logistic regression finds a vector of taxa weights w
that minimizes:
.SIGMA..sub.iln(1+exp(-y.sub.iwx.sub.i))+C.parallel.w.parallel..sub.1
over the training set of samples (x.sub.i, y.sub.i). The constant C
was selected based only on samples from the training set, using
grid search and nested cross-validation.
[0084] The statistical significance of the model in the form of a
p-value was estimated using a permutation test, consisting of
training 10,000 models on data with the class variable randomly
permuted prior to processing, and comparing the distribution of the
10,000 auROC values with the auROC of the original model trained
using unperturbed class variable. Performance of the model on
previously unseen samples was assessed using 200 independent runs
of 5-fold stratified cross-validation, for a total of 1000 training
set--test set pairs. We assessed test set sensitivity, specificity,
and area under ROC curve (auROC), as implemented in the python
scikit-learn package.sup.92. For each of these metrics, we
calculated the average and standard deviation over the 1000
models.
Nucleotide Sequence Accession Numbers
[0085] The sequences of vaginal strains of Sneathia amnii SN35
(accession: NZ_CP011280) and Prevotella timonensis CRIS-5C-B1
(accession: NZ_ADEF01000048) as well as recent submission of BVAB1
S1 (PQV000000000) and TM7-H1 E1 (CP026537) are available in the
Genbank database.
Results
Vaginal Microbiome Profiles Show Preterm Birth-Associated
Trends
[0086] In our longitudinal iHMP study, the Multi-Omic Microbiome
Study--Pregnancy Initiative (MOMS-PI), we enrolled 1,594 pregnant
women from clinics associated with the Research Alliance for
Microbiome Science Registry based at Virginia Commonwealth
University in Virginia and the Global Alliance to Prevent
Prematurity and Stillbirth in Washington. From this cohort, we
analyzed 45 single gestation pregnancies that met criteria for
experiencing spontaneous PTB and 90 single gestation pregnancies
that extended through term (.gtoreq.39 weeks). The TB controls were
matched for age, race, and annual household income for analysis.
Participants were recruited during prenatal visits and samples were
longitudinally collected either by clinicians during a pelvic exam
or by self-sampling. On average, the earliest samples were
collected at 18 weeks gestation, and the mean number of sampling
visits per participant was seven.
[0087] The cohort is predominantly comprised of women of African
ancestry (78%), with a median annual income less than S20,000 and
an average age of 26 years (Table 1).
TABLE-US-00001 TABLE 1 Description of cohort studied in this
project. Preterm Term delivery <37 delivery .gtoreq.39 wks wks
(N = 45) (N = 90) Mean age in years* 26 (5.68) 25.9 (5.43)
Ancestry/Ethnicity African 35 (77.8%) 71 (78.9%) European 6 (13.3%)
13 (14.4%) Hispanic 3 (6.7%) 5 (5.6%) Native American 1 (2.2%) 1
(1.1%) Household Income* <20,000 29 (72.5%) 66 (77.7%)
20,000-59,999 9 (22.5%) 15 (17.6%) 60,000+ 2 (5.0%) 4 (4.7%)
Vaginal Delivery 38 (84.4%) 74 (82.2%) Previous Preterm 14 (31.1%)
10 (11.1%) PPROM 26 (57.8%) 0 (0% *Standard deviation listed in in
parentheses
[0088] Microbiome profiles of the first vaginal samples collected
at study enrollment (FIG. 1) were generated by 16S rRNA. Most
profiles were classified into one of several common community state
types, or vagitypes, dominated by Lactobacillus crispatus,
Lactobacillus iners, other lactobacilli (e.g., Lactobacillus
delbrueckii, Lactobacillus gasseri), A. vaginae, Lachnospiraceae
BVAB1, G. vaginalis, and a complex vagitype with no predominant
taxon (FIG. 1). A significantly smaller proportion of women who
would later experience PTB had a vaginal microbiome characterized
by prevalence of L. crispatus compared with women who would later
deliver at term (p=0.014, FIG. 1b). This result parallels earlier
observations.sup.27 that associate Lactobacillus species with a
more protective state of the vaginal microbiome. A Markov chain
analysis to assess vagitype changes throughout pregnancy reveals
that women who would later deliver prematurely were more likely to
transition to a vagitype characterized by BVAB1 and less likely to
transition to a vagitype characterized by L. crispatus or L.
iners.
[0089] The analysis described above identifies differences in
predominant vagitypes associated with women who will experience
PTB. To identify specific taxa associated with PTB, we compared the
abundance of 26 abundant taxa found in these microbiome profiles
using the earliest samples taken from each of the 45 women who
experienced PTB and the 90 TB controls. We found that overall
diversity was increased in PTB and twelve taxa showed a significant
difference between the PTB and the TB groups. L. crispatus was
significantly reduced in PTB samples, and several other taxa,
including BVAB1, Prevotella cluster 2 and S. amnii, were more
abundant in PTB samples (q<0.05, FIG. 2). Prevotella cluster 2
is comprised of several closely related Prevotella species.sup.48,
and we found that most of these sequences mapped to the reference
sequence for Prevotella timonensis. An analysis using only
enrollment samples that were taken in the first 24 weeks of
pregnancy identified two additional taxa, Megasphaera type 1 and
TM7-H1 (i.e., BVAB-TM7-H1), two bacterial previously associated
with adverse conditions of vaginal health.sup.23, as significantly
increased in PTB. These findings extend those of a previous study
that found BVAB1 and Sneathia species carriage in early and
mid-pregnancy to be associated with spontaneous preterm
birth.sup.36. To our knowledge, this is the first report of an
association of TM7-H1 with PTB.
[0090] We and others.sup.29,49,50 have shown that predominance of
Lactobacillus species tends to be more stable in pregnancy, which
is possibly an evolutionary adaptation by which physiological
changes that occur during pregnancy favor an environment that
reduces the abundance of potentially harmful bacterial species in
the female reproductive tract. Considering that an adverse
pregnancy outcome may be caused by a breakthrough of pathogenic
microbes, this trend suggests that microbiome composition early in
pregnancy may be most useful in prediction of adverse outcomes. We
examined the longitudinal trends of several of the taxa
significantly correlated, both positively and negatively, with PTB.
FIG. 3 shows the results of a generalized additive mixed effect
model (GAMM) incorporating BMI, ethnicity (i.e., African or
European ancestry), pregnancy outcome (i.e, PTB, TB), a smoother
for gestational age, which captures the major trends while leaving
out noise and fine-scale structures in the data, and a random
subject effect to longitudinally model log-transformed relative
abundances of vaginally relevant taxa. As we.sup.32 and
others.sup.51 have noted, the vaginal microbiome profiles of women
of African and European ancestry differ significantly, and we
therefore stratified the analysis by ancestry. In this analysis,
BVAB1, G. vaginalis, L. crispatus, P. amnii, Prevotella cluster 2,
and S. amnii were significantly different in women who deliver
preterm or term. Moreover, based on GAMM fit, women of African
ancestry who deliver preterm experience significant decreases, over
the term of pregnancy, in prevalence of A. vaginae (p=0.005), BVAB1
(p=0.0001), G. vaginalis (p=0.0001), P. amnii (p=0.0003), S. amnii
(p=0.017) and TM7-H1 (p=0.001). Women of African ancestry who
delivered full term exhibited fewer changes in the modeled taxa
throughout pregnancy, although decreases in prevalence of A.
vaginae (p=0.0001) and G. vaginalis (p=0.0001), and an increase in
L. iners (p=0.015) were observed. Women of European ancestry
exhibited microbiome profiles with greater apparent stability
during pregnancy, although an increase in prevalence of G.
vaginalis (p=0.002) was noted for women who delivered preterm.
Women of African ancestry have a significantly increased risk of
PTB compared to women of European ancestry. Thus, we may expect
that with a case-control study design, carriage of taxa with
intermediate risk, such as the phylotypes of G. vaginalis, may be
associated with PTB in a lower-risk cohort, but TB in a higher-risk
cohort. We previously reported.sup.32 that carriage of L.
crispatus, which is negatively associated with PTB, is more
prevalent in women of European ancestry, and BVAB1, which is
positively associated with PTB, is more common in women of African
ancestry. Thus, our findings are consistent with a proposed
framework in which there exists a comprehensive spectrum of vaginal
microbiome states linked to risk for preterm birth. Note that
relative abundance of bacterial taxa associated with PTB is
generally quite low in the early stages of pregnancy in women of
African ancestry who experience TB, as well most women of European
ancestry, and significant decreases may therefore be difficult to
detect (FIG. 3).
[0091] Vaginal samples from each of the participants were subjected
to metagenomic sequencing and a subset of those samples that were
collected between 14 and 27 weeks gestation were also subjected to
metatranscriptomic analysis as outlined in the Methods. A pathway
analysis identified a relatively conserved functional and metabolic
potential of the microbial communities, independent of vagitype,
across the samples. However, the proportional transcriptional
activity devoted to each pathway varied. For example, L.
crispatus-dominated samples had significantly higher proportional
transcriptional activity of UDP-N-acetyl-D-glucosamine
biosynthesis, which is involved in production of peptidoglycan.
Peptidoglycan is one of the best described microbe-associated
molecular patterns (MAMPs) involved in the modulaton of host
cytokine production via Toll-like receptor signaling. Conversely,
the proportional transcriptional activity of genes classified to
the pyruvate fermentation to acetate and lactate II and the
non-oxidative branch of the pentose phosphate pathway was lower in
L. crispatus-dominated samples. This finding is consistent with
previous reports of reduced levels of lactic acid and increased
concentrations of short-chain fatty acids (SCFA) (e.g., acetate,
propionate, butyrate, and succinate) in vaginal samples of women
with bacterial vaginosis. Intriguingly, SCFAs have been suggested
to reduce anti-microbial activity and promote proinflammatory
cytokines in the vaginal environment.sup.52.
Bacterial Taxa Associated with PTB Encode and Express Potential
Virulence Factors
[0092] Metagenomic sequence data were assembled to characterize the
genomes of BVAB1 and TM7-H1, respectively. To our knowledge, these
taxa have not been previously cultivated or characterized beyond
their 16S rRNA sequences. We examined these genomes along with the
available genomes of S. amnii SN35 (NZ_CP011280), which we
previously reported.sup.53, and P. timonensis CRIS-5C-B1
(NZ_ADEF01000048). The genome sizes were .about.0.72 Mb for TM7-H1
(CP026537), .about.1.34 Mb for S. amnii (CP011280.1), .about.1.45
Mb for BVAB1 (PQV000000000), and .about.2.8 Mb for P. timonensis
(NBAX01000001). BVAB1, P. timonensis, and S. amnii are classified
to the Lachnospiraceae, Leptotrichiaceae, and Prevotellaceae
families, respectively. TM7-H1 falls into the Candidatus
Saccharibacteria phylum and exhibits .about.66% nucleotide identity
to the recently described oral TM7x (NZ_CP007496). TM7-H1 encodes a
putative alpha-amylase and is predicted to be able to utilize
glycogen as a carbon source. Like TM7x.sup.54, TM7-H1 lacks de novo
biosynthetic capabilities for essential amino acids and likely
depends on other organisms in the vaginal environment for survival.
TM7x has been characterized as an obligate, parasitic epibiont; it
is unknown whether TM7-H1 similarly lives on the surface of another
bacterial species in the vaginal environment. Using these reference
genomes, we mapped taxon-specific transcripts from
metatranscriptomic data from the vaginal samples of PTB and TB
controls and observed that, although there was some variation in
the activities of several metabolic and signaling pathways, the
overall transcriptional profiles of each of these taxa were largely
conserved across both PTB cases and TB controls.
[0093] We looked at broad classes of genes with possible roles in
virulence and defense. Genes annotated with putative roles in
antibiotic resistance, resistance to toxic compounds and oxidative
stress were identified in all four genomes. Other putative defenses
were variable; for example, 57 genes predicted to be involved in
motility were identified in BVAB1, many of which were transcribed
at high levels in vaginal samples. There was no obvious enhancement
of expression levels of the virulence genes in women who would
later experience PTB, a largely expected result since expression
levels are likely controlled by general conditions of the vaginal
environment. We further performed a metabolic reconstruction and
modeling of TM7-H1 and BVAB1. We identified 243 metabolic reactions
in TM7-H1 and and 421 metabolic reactions in BVAB1. Both of the
organisms are predicted to have the ability to produce pyruvate,
acetate, L-lactate and propionate; BVAB1 encodes additional
pathways for production of acetaldehyde, D-lactate formate, and
acetyl-CoA. Neither is predicted to take part in the TCA cycle, and
TM7-H1 completely lacks genes related to butyrate metabolism. As
described above, production of SCFAs has been linked to a
proinflammatory state.sup.52, with possible implications for
disease. As such, these metabolic models lay the foundations for
understanding possible mechanisms by which these bacteria impact
pregnancy. Taken together, identification of these virulence genes
and factors in these four bacterial strains is consistent with our
finding that they are associated with PTB. More importantly,
characterization of the genomes of these strains and identification
of likely determinants of pathogenicity represents an important
step toward understanding the mechanisms by which components of the
vaginal microbiome mediate or cause PTB.
Host Cytokine Expression in PTB
[0094] To examine the roles of cytokines in the progression of
pregnancy to PTB, we measured cytokine levels in vaginal swab
samples. Using the cytokine data from nine (IL-1b, IL-6, IL-8,
Eotaxin, TNF-.alpha., IL-17A, MIP-1b, IP-10, RANTES) of the 27
examined, and data from the 16S rRNA taxonomic surveys, both log
transformed, we performed an integrative sparse canonical
correlation analysis to assess the correlation between bacterial
taxa and cytokine levels (FIG. 4). For each participant, the
observation corresponding to the earliest gestational age per
trimester was used in the analysis. In women who delivered at term
(FIG. 4a), we observed a negative correlation between L. crispatus
and the taxa associated with dysbiosis and PTB (e.g., G. vaginalis,
Prevotella cluster 2, S. amnii, and to a lesser extent TM7-H1), as
well as with the analyzed cytokines. The analyzed cytokines, with
the exception of IP-10 are largely proinflammatory in function,
were positively correlated both with each other and with taxa
associated with dysbiosis and PTB. In contrast, we observed that
IP-10 was positively associated with L. iners, an association that
has been reported by others.sup.55, and negatively associated with
both L. crispatus and taxa associated with dysbiosis.
[0095] There were notable differences in the taxa-cytokine
correlations in women who went on to experience PTB. The
proinflammatory cytokines (e.g., IL-1b) and dysbiotic taxa (e.g. A.
vaginae, G. vaginalis, and Megasphaera type 1) tend to form a
tighter cluster. In contrast, IP-10 did not cluster with L. iners
in the PTB cohort and BVAB1, which was not selected as a feature by
the analysis in the TB group, was negatively correlated with IP-10
in PTB samples. These observations generally support the concept
that bacterial taxa in women who experience PTB (e.g., Prevotella
cluster 2, S. amnii and TM7-H1, among others) are positively
correlated with proinflammatory cytokines, and negatively
correlated with taxa (e.g., L. crispatus) that are negatively
correlated with PTB.
Predictive Model for PTB
[0096] Early prediction of risk for PTB is critical for the
development of new strategies for prevention and intervention.
Using a set of 31 PTB and 59 TB subjects that had samples collected
early (6-24 weeks gestational age) in pregnancy, we developed a
predictive model for PTB. Model construction involved selecting
taxa that are differentially represented in the PTB and TB cohorts
as assessed using the Mann-Whitney U test (FIG. 5a), and assigning
weights to these taxa using L.sub.1-regularized logistic
regression. The resulting model incorporates four taxa: S. amnii,
BVAB1, Prevotella cluster 2, and TM7-H1, which are all positively
correlated to PTB (FIG. 5a). The model is significant (p=0.0024)
and has expected sensitivity of 76.+-.17% (mean.+-.SD), specificity
of 74.+-.13%, and an area under the ROC curve of 0.769.+-.0.108 for
samples not used during training. Thus, this modeling strategy
represents a promising approach to utilization of microbiome data
obtained early in pregnancy to identify pregnancies with higher
risk of PTB for possible prophylaxis.
Conclusions
[0097] Our comparison of the vagitypes in 45 women who experienced
PTB to 90 women who experience TB revealed a significant difference
in the proportion of samples characterized by predominance of L.
crispatus. Consistent with this observation, we identified 12 taxa
that were significantly associated with PTB in this cohort. L.
crispatus, which is thought to play a generally protective role in
the female reproductive tract, was negatively associated with PTB;
the other 11 taxa, many of which have been implicated in vaginal
dysbiosis and bacterial vaginosis, were positively correlated with
PTB. BVAB1, Prevotella cluster 2, Sneathia amnii, and TM7-H1, were
consistently identified as relevant to PTB in multiple analyses. A
network analysis of these four taxa (FIG. 5b) show them to be
positively correlated with taxa associated with vaginal dysbiosis.
At the selected threshold, Prevotella cluster 2 was the only taxon
that had negative correlations with Lactobacillus species, which is
intruiging given that Prevotella species have been reported to be
associated with PTB in both low-risk and high-risk cohorts of women
of European and African ancestry.sup.39. We further found that
Prevotella species, including P. timonensis, showed higher relative
abundance in rectal samples compared to vaginal samples, consistent
with the hypothesis that a recto-vaginal pathway may play a role
for some key taxa that are associated with preterm birth. These
relationships indicate that neither the vagitype nor a small number
of indicator bacteria are sufficient to fully characterize PTB
risk, but rather complex interactions between bacterial communities
and the host are important.
[0098] In women of African ancestry, taxa significantly associated
with PTB were more highly correlated early in pregnancy. This
finding is consistent with previous observations that pregnancy
tends to be associated with reduced carriage of bacterial
vaginosis-associated organisms. Our longitudinal modeling supported
this concept in that taxa associated with PTB tended to decrease in
abundance in the vaginal environment throughout pregnancy. We
observed that women of African ancestry who experienced PTB often
exhibited an initial decrease in prevalence of these taxa followed
by a reappearance in the third trimester. This finding is
consistent with a proposed model in which carriage of one or more
of taxa associated with PTB in early pregnancy confers an increased
risk for a subsequent breakthrough event that leads to infection
and inflammatory processes that induce premature delivery.
[0099] Because the composition of the vaginal microbiome in early
pregnancy may be more relevant to PTB, we explored a predictive
model for PTB using taxonomic data from early pregnancy vaginal
samples. The model focused on the three taxa that consistently
showed an association with PTB (i.e., BVAB1, Prevotella cluster 2,
S. amnii) and TM7-H1, which was not prominent in our initial
analyses, probably because it is generally of lower abundance and
tends to peak in very early pregnancy. Inclusion of TM7-H1 provided
a significant enhancement to the model, which has promise for the
early prediction of PTB with mean sensitivity and specificity of
.about.75%.
[0100] The data suggest that the four taxa used on our predictive
model (i.e., BVAB1, Prevotella cluster 2, S. amnii and TM7-H1) have
roles in causation of PTB. We previously characterized the genome
of S. amnii, identifying several potential cytotoxins, and showed
that cultured bacteria kill eukaryotic cells in vitro.sup.53.
Moreover, analysis of our metagenomic genome assemblies of BVAB1
and TM7 and an available genome of P. timonensis identified
multiple potential virulence factors, antibiotic resistance genes,
episomal elements, and genes encoding proteins associated with
cellular invasion and intracellular existence. These factors can
now be readily genetically manipulated and tested for activity in
heterologous systems. Our analysis of cytokine expression also
represents an attempt to incorporate causal and mechanistic insight
into the relationship between the vaginal microbiome and risk of
PTB. Labor is associated with proinflammatory cytokine expression,
and premature labor can be induced by inflammatory responses. Our
analysis is consistent with previous findings and shows that
bacterial taxa generally associated with dysbiosis and bacterial
vaginosis are highly correlated with expression of pro-inflammatory
cytokines, which may play a role in induction of labor. We also
observed that vaginal IP-10 levels were inversely correlated with
BVAB1 in PTB, inversely correlated with L. crispatus in TB and
positively correlated with L. iners in TB cohort, suggesting that
there exist complex host-microbiome interactions in pregnancy.
Previous studies.sup.12 disclosed the relative contributions of
maternal and fetal genetics to PTB, and the impact of bacterial
vaginosis, implicating gene-environment interactions that modulate
the maternal and fetal immunological and inflammatory cascades,
resulting in early labor and delivery. In apparent contradiction to
the conclusion that bacteria play a role in triggering PTB,
treatment of bacterial vaginosis and vaginal dysbiosis with the
antibiotics, metronidazole or clindamycin, failed to prevent
PTB.sup.56,57. This may not be surprising, since it is becoming
apparent that multiple bacterial taxa of widely different phylogeny
are positively associated with PTB and that they may interact with
each other and the host in complex biofilm-formations. Thus, a
single antibiotic may not target all relevant taxa and might in
fact clear the way for pathogenic culprits by eliminating their
healthier competitors. The genomes of these bacteria may harbor
mechanistic clues such as genes associated with antibiotic
resistance, defense, and genes encoding putative surface proteins
that may modulate microbe-microbe and microbe-host interactions.
Further, more in-depth analysis of metabolic models may yield
insight into how these taxa rely on each other and compete for
resources within the vaginal environment. The findings described
herein provide new insights and methods which can be used to
prospectively assess risk of PTB and create strategies for
prophylaxis treatment.
REFERENCES
[0101] 1. WHO|Preterm birth. WHO Available at:
http://www.who.int/mediacentre/factsheets/fs363/en/. (Accessed: 1st
November 2017) [0102] 2. Blencowe, H. et al. Born Too Soon: The
global epidemiology of 15 million preterm births. Reprod. Health
10, S2 (2013). [0103] 3. Marret, S. et al. Neonatal and 5-year
Outcomes After Birth at 30-34 Weeks of Gestation. Obstet. Gynecol.
110, 72-80 (2007). [0104] 4. Wolke, D., Eryigit-Madzwamuse, S.
& Gutbrod, T. Very preterm/very low birthweight infants'
attachment: infant and maternal characteristics. Arch. Dis.
Child.--Fetal Neonatal Ed. 99, F70-F75 (2014). [0105] 5. Verrips,
G. et al. Long term follow-up of health-related quality of life in
young adults born very preterm or with a very low birth weight.
Health Qual. Life Outcomes 10, 49 (2012). [0106] 6. Wolke, D. et
al. Self and Parent Perspectives on Health-Related Quality of Life
of Adolescents Born Very Preterm. J. Pediatr. 163, 1020-1026.e2
(2013). [0107] 7. Simms, V. et al. Mathematics difficulties in
extremely preterm children: evidence of a specific deficit in basic
mathematics processing. Pediatr. Res. 73, 236-244 (2013). [0108] 8.
Murray, C. J. L. et al. Disability-adjusted life years (DALYs) for
291 diseases and injuries in 21 regions, 1990-2010: a systematic
analysis for the Global Burden of Disease Study 2010. The Lancet
380, 2197-2223 (2012). [0109] 9. Blencowe, H., Lawn, J. E.,
Vazquez, T., Fielder, A. & Gilbert, C. Preterm-associated
visual impairment and estimates of retinopathy of prematurity at
regional and global levels for 2010. Pediatr. Res. 74, 35-49
(2013). [0110] 10. Manuck, T. A. Racial and ethnic differences in
preterm birth: A complex, multifactorial problem. Semin. Perinatol.
(2017). doi:10.1053/j.semperi.2017.08.010 [0111] 11. Behrman, R.
E., Butler, A. S. & Outcomes, I. of M. (US) C. on U. P. B. and
A. H. Societal Costs of Preterm Birth. (National Academies Press
(US), 2007). [0112] 12. York, T. P., Eaves, L. J., Neale, M. C.
& Strauss, J. F. The contribution of genetic and environmental
factors to the duration of pregnancy. Am. J. Obstet. Gynecol. 210,
398-405 (2014). [0113] 13. Preterm Birth: Causes, Consequences, and
Prevention. (National Academies Press (US), 2007). [0114] 14.
Goldenberg, R. L., Culhane, J. F., Jams, J. D. & Romero, R.
Epidemiology and causes of preterm birth. Lancet Lond. Engl. 371,
75-84 (2008). [0115] 15. Donders, G. G. et al. Predictive value for
preterm birth of abnormal vaginal flora, bacterial vaginosis and
aerobic vaginitis during the first trimester of pregnancy. BJOG
Int. J. Obstet. Gynaecol. 116, 1315-1324 (2009). [0116] 16.
Martius, J. et al. Relationships of vaginal Lactobacillus species,
cervical Chlamydia trachomatis, and bacterial vaginosis to preterm
birth. Obstet. Gynecol. 71, 89-95 (1988). [0117] 17. What are the
risk factors for preterm labor and birth? Available at:
https://www.nichd.nih.gov/health/topics/preterm/conditioninfo/Pages/who_r-
isk.aspx. (Accessed: 1st November 2017) [0118] 18. Romero, R., Dey,
S. K. & Fisher, S. J. Preterm Labor: One Syndrome, Many Causes.
Science 345, 760-765 (2014). [0119] 19. Cobb, C. M. et al. The oral
microbiome and adverse pregnancy outcomes. Int. J. Womens Health 9,
551-559 (2017). [0120] 20. Pretorius, C., Jagatt, A. & Lamont,
R. F. The relationship between periodontal disease, bacterial
vaginosis, and preterm birth. J. Perinat. Med. 35, 93-99 (2007).
[0121] 21. Parihar, A. S. et al. Periodontal Disease: A Possible
Risk-Factor for Adverse Pregnancy Outcome. J. Int. Oral Health JIOH
7, 137-142 (2015). [0122] 22. Structure, function and diversity of
the healthy human microbiome. Nature 486, 207-214 (2012). [0123]
23. Fredricks, D. N., Fiedler, T. L., Thomas, K. K., Oakley, B. B.
& Marrazzo, J. M. Targeted PCR for Detection of Vaginal
Bacteria Associated with Bacterial Vaginosis. J. Clin. Microbiol.
45, 3270-3276 (2007). [0124] 24. Sobel, J. D. Bacterial vaginosis.
Annu. Rev. Med. 51, 349-356 (2000). [0125] 25. Bradshaw, C. S.
& Sobel, J. D. Current Treatment of Bacterial
Vaginosis--Limitations and Need for Innovation. J. Infect. Dis.
214, S14-S20 (2016). [0126] 26. Chavoustie, S. E. et al. Experts
explore the state of bacterial vaginosis and the unmet needs facing
women and providers. Int. J. Gynecol. Obstet. (2017).
doi:10.1002/ijgo.12114 [0127] 27. Ma, B., Forney, L. J. &
Ravel, J. The vaginal microbiome: rethinking health and diseases.
Annu. Rev. Microbiol. 66, 371-389 (2012). [0128] 28. Hickey, R. J.,
Zhou, X., Pierson, J. D., Ravel, J. & Forney, L. J.
Understanding vaginal microbiome complexity from an ecological
perspective. Transl. Res. J. Lab. Clin. Med. 160, 267-282 (2012).
[0129] 29. MacIntyre, D. A. et al. The vaginal microbiome during
pregnancy and the postpartum period in a European population. Sci.
Rep. 5, 8988 (2015). [0130] 30. Ravel, J. et al. Vaginal microbiome
of reproductive-age women. Proc. Natl. Acad. Sci. U.S.A 108 Suppl
1, 4680-4687 (2011). [0131] 31. Martin, D. H. & Marrazzo, J. M.
The Vaginal Microbiome: Current Understanding and Future
Directions. J. Infect. Dis. 214, S36-S41 (2016). [0132] 32.
Fettweis, J. M. et al. Differences in vaginal microbiome in African
American women versus women of European ancestry. Microbiol. Read.
Engl. 160, 2272-2282 (2014). [0133] 33. Zhou, X. et al. Differences
in the composition of vaginal microbial communities found in
healthy Caucasian and black women. ISME J. 1, 121-133 (2007).
[0134] 34. Hyman, R. W. et al. Diversity of the Vaginal Microbiome
Correlates With Preterm Birth. Reprod. Sci. 21, 32-40 (2014).
[0135] 35. Beamer, M. A. et al. Bacterial species colonizing the
vagina of healthy women are not associated with race. Anaerobe 45,
40-43 (2017). [0136] 36. Nelson, D. B. et al. Early Pregnancy
Changes in Bacterial Vaginosis-Associated Bacteria and Preterm
Delivery. Paediatr. Perinat. Epidemiol. 28, 88-96 (2014). [0137]
37. Romero, R. et al. The vaginal microbiota of pregnant women who
subsequently have spontaneous preterm labor and delivery and those
with a normal delivery at term. Microbiome 2, 18 (2014). [0138] 38.
DiGiulio, D. B. et al. Temporal and spatial variation of the human
microbiota during pregnancy. Proc. Natl. Acad. Sci. U.S.A 112,
11060-11065 (2015). [0139] 39. Callahan, B. J. et al. Replication
and refinement of a vaginal microbial signature of preterm birth in
two racially distinct cohorts of US women. Proc. Natl. Acad. Sci.
U.S.A 114, 9966-9971 (2017). [0140] 40. Brown, R. et al. Role of
the vaginal microbiome in preterm prelabour rupture of the
membranes: an observational study. The Lancet 387, S22 (2016).
[0141] 41. Nelson, D. B., Shin, H., Wu, J. & Dominguez-Bello,
M. G. The Gestational Vaginal Microbiome and Spontaneous Preterm
Birth among Nulliparous African American Women. Am. J. Perinatol.
33, 887-893 (2016). [0142] 42. Stout, M. J. et al. Early pregnancy
vaginal microbiome trends and preterm birth. Am. J. Obstet.
Gynecol. 217, 356.e1-356.e18 (2017). [0143] 43. DiGiulio, D. B.
Diversity of microbes in amniotic fluid. Semin. Fetal. Neonatal
Med. 17, 2-11 (2012). [0144] 44. Goldenberg, R. L. et al. The
Alabama Preterm Birth Study: Umbilical cord blood Ureaplasma
urealyticum and Mycoplasma hominis cultures in very preterm newborn
infants. Am. J. Obstet. Gynecol. 198, 43.e1-43.e5 (2008). [0145]
45. Han, Y. W., Shen, T., Chung, P., Buhimschi, I. A. &
Buhimschi, C. S. Uncultivated bacteria as etiologic agents of
intra-amniotic inflammation leading to preterm birth. J. Clin.
[0146] Microbiol. 47, 38-47 (2009). [0147] 46. Kindinger, L. M. et
al. The interaction between vaginal microbiota, cervical length,
and vaginal progesterone treatment for preterm birth risk.
Microbiome 5, 6-6 (2017). [0148] 47. The Integrative Human
Microbiome Project: Dynamic Analysis of Microbiome-Host Omics
Profiles during Periods of Human Health and Disease. Cell Host
Microbe 16, 276-289 (2014). [0149] 48. Fettweis, J. M. et al.
Species-level classification of the vaginal microbiome. BMC
Genomics 13 Suppl 8, S17 (2012). [0150] 49. Romero, R. et al. The
composition and stability of the vaginal microbiota of normal
pregnant women is different from that of non-pregnant women.
Microbiome 2, 4 (2014). [0151] 50. Walther-Antonio, M. R. S. et al.
Pregnancy's stronghold on the vaginal microbiome. PloS One 9,
e98514 (2014). [0152] 51. Borgdorff, H. et al. The association
between ethnicity and vaginal microbiota composition in Amsterdam,
the Netherlands. PloS One 12, e0181135 (2017). [0153] 52. Aldunate,
M. et al. Antimicrobial and immune modulatory effects of lactic
acid and short chain fatty acids produced by vaginal microbiota
associated with eubiosis and bacterial vaginosis. Front. Physiol.
6, 164 (2015). [0154] 53. Harwich, M. D., Jr et al. Genomic
sequence analysis and characterization of Sneathia amnii sp. nov.
BMC Genomics 13 Suppl 8, S4 (2012). [0155] 54. He, X. et al.
Cultivation of a human-associated TM7 phylotype reveals a reduced
genome and epibiotic parasitic lifestyle. Proc. Natl. Acad. Sci.
U.S.A 112, 244-249 (2015). [0156] 55. Jespers, V. et al. A
longitudinal analysis of the vaginal microbiota and vaginal immune
mediators in women from sub-Saharan Africa. Sci. Rep. 7, 11974
(2017). [0157] 56. Haahr, T. et al. Treatment of bacterial
vaginosis in pregnancy in order to reduce the risk of spontaneous
preterm delivery--a clinical recommendation. Acta Obstet. Gynecol.
Scand. 95, 850-860 (2016). [0158] 57. Myrhaug, H. T., Brurberg, K.
G., Kirkehei, I. & Reinar, L. M. Treatment of Pregnant Women
with Asymptomatic Bacterial Vaginosis with Clindamycin. (Knowledge
Centre for the Health Services at The Norwegian Institute of Public
Health (NIPH), 2010). [0159] 58. Forney, L. J. et al. Comparison of
Self-Collected and Physician-Collected Vaginal Swabs for Microbiome
Analysis. J. Clin. Microbiol. 48, 1741-1748 (2010). [0160] 59.
Parikh, H. I., Koparde, V. N., Bradley, S. P., Buck, G. A. &
Sheth, N. U. MeFiT: merging and filtering tool for illumina
paired-end reads for 16S rRNA amplicon sequencing. BMC
Bioinformatics 17, 491 (2016). [0161] 60. Edgar, R. C., Haas, B.
J., Clemente, J. C., Quince, C. & Knight, R. UCHIME improves
sensitivity and speed of chimera detection. Bioinforma. Oxf. Engl.
27, 2194-2200 (2011). [0162] 61. Edgar, R. C. Search and clustering
orders of magnitude faster than BLAST. Bioinforma. Oxf. Engl. 26,
2460-2461 (2010). [0163] 62. Yarza, P. et al. Update of the
All-Species Living Tree Project based on 16S and 23S rRNA sequence
analyses. Syst. Appl. Microbiol. 33, 291-299 (2010). [0164] 63.
Chen, T. et al. The Human Oral Microbiome Database: a web
accessible resource for investigating oral microbe taxonomic and
genomic information. Database J. Biol. Databases Curation 2010,
baq013 (2010). [0165] 64. Hartmann, M., Howes, C. G., Abarenkov,
K., Mohn, W. W. & Nilsson, H. V-Xtractor: An open-source,
high-throughput software tool to identify and extract hypervariable
regions of small subunit (16 S/18 S) ribosomal RNA gene sequences.
J. Microbiol. Methods 83, 250-3 (2010). [0166] 65. Lindgreen, S.
AdapterRemoval: easy cleaning of next-generation sequencing reads.
BMC Res. Notes 5, 337 (2012). [0167] 66. Koparde, V. N., Parikh, H.
I., Bradley, S. P. & Sheth, N. U. MEEPTOOLS: a maximum expected
error based FASTQ read filtering and trimming toolkit. Int. J.
Comput. Biol. Drug Des. 10, 237-247 (2017). [0168] 67. Li, H. &
Durbin, R. Fast and accurate long-read alignment with
Burrows-Wheeler transform. Bioinforma. Oxf. Engl. 26, 589-595
(2010). [0169] 68. Alves, J. M. P. & Buck, G. A. Automated
system for gene annotation and metabolic pathway reconstruction
using general sequence databases. Chem. Biodivers. 4, 2593-2602
(2007). [0170] 69. Langille, M. G. I. et al. Predictive functional
profiling of microbial communities using 16S rRNA marker gene
sequences. Nat. Biotechnol. 31, 814-821 (2013). [0171] 70.
Kaminski, J. et al. High-Specificity Targeted Functional Profiling
in Microbial Communities with ShortBRED. PLOS Comput. Biol. 11,
e1004557 (2015). [0172] 71. Altschul, S. F., Gish, W., Miller, W.,
Myers, E. W. & Lipman, D. J. Basic local alignment search tool.
J. Mol. Biol. 215, 403-410 (1990). [0173] 72. Bankevich, A. et al.
SPAdes: a new genome assembly algorithm and its applications to
single-cell sequencing. J. Comput. Biol. J. Comput. Mol. Cell Biol.
19, 455-477 (2012). [0174] 73. Nurk, S., Meleshko, D.,
Korobeynikov, A. & Pevzner, P. A. metaSPAdes: a new versatile
metagenomic assembler. Genome Res. 27, 824-834 (2017). [0175] 74.
Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with
Bowtie 2. Nat. Methods 9, 357-359 (2012). [0176] 75. Lin, H.-H.
& Liao, Y.-C. Accurate binning of metagenomic contigs via
automated clustering sequences using information of genomic
signatures and marker genes. Sci. Rep. 6, 24175 (2016). [0177] 76.
Seemann, T. Prokka: rapid prokaryotic genome annotation.
Bioinforma. Oxf. Engl. 30, 2068-2069 (2014). [0178] 77. Lowe, T. M.
& Eddy, S. R. tRNAscan-SE: a program for improved detection of
transfer RNA genes in genomic sequence. Nucleic Acids Res. 25,
955-964 (1997). [0179] 78. Lagesen, K. et al. RNAmmer: consistent
and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 35,
3100-3108 (2007). [0180] 79. Aziz, R. K. et al. The RAST Server:
rapid annotations using subsystems technology. BMC Genomics 9, 75
(2008). [0181] 80. Scholz, M. et al. Strain-level microbial
epidemiology and population genomics from shotgun metagenomics.
Nat. Methods 13, 435-438 (2016). [0182] 81. Brooks, J. P. et al.
Changes in vaginal community state types reflect major shifts in
the microbiome. Microb. Ecol. Health Dis. 28, 1303265 (2017).
[0183] 82. Benjamini, Y. & Hochberg, Y. Controlling the False
Discovery Rate: A Practical and Powerful Approach to Multiple
Testing. J. R. Stat. Soc. Ser. B Methodol. 57, 289-300 (1995).
[0184] 83. Lin, X. & Zhang, D. Inference in generalized
additive mixed models by using smoothing splines. J. R. Stat. Soc.
Ser. B Stat. Methodol. 61, 381-400 (1999). [0185] 84. Harville, D.
Maximum likelihood approaches to variance component estimation and
to related problems. J. Am. Stat. Assoc. 72, 320-338 (1977). [0186]
85. Parkhomenko, E., Tritchler, D. & Beyene, J. Sparse
canonical correlation analysis with application to genomic data
integration. Stat. Appl. Genet. Mol. Biol. 8, Article 1 (2009).
[0187] 86. Witten, D. M., Tibshirani, R. & Hastie, T. A
penalized matrix decomposition, with applications to sparse
principal components and canonical correlation analysis.
Biostatistics 10, 515-534 (2009). [0188] 87. Gonzalez, I., Cao,
K.-A. L., Davis, M. J. & Dejean, S. Visualising associations
between paired `omics` data sets. BioData Min. 5, 19 (2012). [0189]
88. Rohart, F., Gautier, B., Singh, A. & L Cao, K.-A. mixOmics:
An R package for `omics feature selection and multiple data
integration. PLoS Comput. Biol. 13, e1005752 (2017). [0190] 89.
Ban, Y., An, L. & Jiang, H. Investigating microbial
co-occurrence patterns based on metagenomic compositional data.
Bioinforma. Oxf. Engl. 31, 3322-3329 (2015). [0191] 90. Bastian,
M., Heymann, S. & Jacomy, M. Gephi: an open source software for
exploring and manipulating networks. Int. AAAI Conf. Weblogs Soc.
Media (2009). [0192] 91. Ng, A. Y. Feature Selection, L1 vs. L2
Regularization, and Rotational Invariance. in Proceedings of the
Twenty-first International Conference on Machine Learning 78--(ACM,
2004). doi:10.1145/1015330.1015435 [0193] 92. Pedregosa, F. et al.
Scikit-learn: Machine learning in Python. J. Mach. Learn. Res. 12,
2825-2830 (2011).
[0194] While the invention has been described in terms of its
preferred embodiments, those skilled in the art will recognize that
the invention can be practiced with modification within the spirit
and scope of the appended claims. Accordingly, the present
invention should not be limited to the embodiments as described
above, but should further include all modifications and equivalents
thereof within the spirit and scope of the description provided
herein.
Sequence CWU 1
1
261439DNACandidatus Saccharibacteria genomosp. TM7-H1 1gatgaacgct
ggcggcatgc ctaacacatg caagtcgatc ggtaaggccc ttcgggtaca 60cgagaggcgg
acggctgagt aacgcgtggg aacgcaccct acactgaggg ataagatacc
120gaaaggtgtt ctaataccgc atacggtctt cggattaaag tcttcggacg
gtgaaggagc 180ggcccgcgtc atattaggta gttggtgggg taatggccta
ccaagccgat gatgtgtagc 240tggtctgaga ggatgatcag ccagactgga
actgagaacg gtccagactc ctacgggagg 300cagcagtgag gaatattcca
caatgggcga aagcctgatg gagcaatgcc gcgtgcagga 360tgaaggccct
cgggtcgtaa actgctttta agagaagaat atgacggtaa ctaatgaata
420agggacggct aactacgtg 4392480DNAClostridiales genomosp. BVAB1
2gatgaacgtt ggcggcgtgc ttaacacatg caagtcgaac gaagtgctac gacggaagtt
60ttcggacgga agatgtagtt acttagtggc ggacgggtga gtaacgcgtg gggaacctgc
120cctgtaccgg gggatagcag ccggaaacgg ctgataatac cgcataagcg
cacaaatgtc 180gcatgacatg gtgtgaaaaa ctccggtggt ataggatgga
cccgcgtctg attagccggt 240tggtggggta aaagcctacc aaagcgaaga
tcagtagccg agttgagaga ctgaccggcc 300acattgggac tgagacacgg
cccagactcc tacgggaggc agcagtgggg aatattgcac 360aatgggcgaa
agcctgatgc agcgacgccg cgtgagcgaa gaagtatttc ggtatgtaaa
420gctctatcag aagggaagaa aatgacggta ccttactaag aagctccggc
taaatacgtg 4803463DNASneathia amnii 3gataaacgct gacagaatgc
ttaacacatg caagtctatg aggaagttta gcttgctaaa 60tggactcatg gcggacgggt
gagtaacgcg taaagaactt gccctttaga ctgggataac 120agagggaaac
ttctgataat actggataag ttagtatatc gcatgatatg caaatgaaag
180ctacggcact aaaggagagc tttgcgtcct attagctagt tggtaaggta
agagcttacc 240aaggcgatga taggtagccg gcctgagagg gtggacggcc
acaaggggac tgagatacgg 300cccttactcc tacgggaggc agcagtgggg
aatattggac aatggaggga actctgatcc 360agcaattctg tgtgtgtgaa
gaaggtttta ggactgtaaa acacttttag tagggaagaa 420aaaaatgacg
gtacctacag aagaagcaac ggctaaatac gtg 4634490DNAPrevotella OTU46
4gatgaacgct agctacaggc ttaacacatg caagtcgcag ggtaacgtga gggaagcttg
60ctttccttga cgacgactgg cgcacgggtg agtaacgcgt atccaacctt cccatgacca
120cgggataacc cgttgaaaga cggcctaata ccgtatgacg tcgtttgccg
acatcgaaag 180acgaccaaag gtttggcggt gatggatggg gatgcgtctg
attagcttgt tggcggggta 240acggcccacc aaggcgacga tcagtagggg
ttctgagagg aaggtccccc acattggaac 300tgagacacgg tccaaactcc
tacgggaggc agcagtgagg aatattggtc aatgggcgag 360agcctgaacc
agccaagtag cgtgcaggat gacggcccta tgggttgtaa actgctttta
420cgcggggata aagtgcgtga cgtgtctcgc attgcaggta ccgcgtgaat
aaggaccggc 480taattccgtg 4905490DNAPrevotella buccalis 5gatgaacgct
agctacaggc ttaacacatg caagtcgcag ggtaacgtga gggaagcttg 60cttcccttga
cgacgactgg cgcacgggtg agtaacgcgt atccaacctt cccatgacca
120cgggataacc cgttgaaaga cggactaata ccgtatgacg tcgtttgctg
acatcaaata 180acgattaaag gtttagcggt gatggatggg gatgcgtctg
attagcttgt tggcggggta 240acggcccacc aaggcgacga tcagtagggg
ttctgagagg aaggtccccc acattggaac 300tgagacacgg tccaaactcc
tacgggaggc agcagtgagg aatattggtc aatgggcgag 360agcctgaacc
agccaagtag cgtgcaggat gacggcccta tgggttgtaa actgctttta
420tgcggggata aagtgcgcga cgtgtcgtgc attgcaggta ccgcatgaat
aaggaccggc 480taattccgtg 4906490DNAPrevotella timonensis
6gatgaacgct agctacaggc ttaacacatg caagtcgcag ggtaacatga ggaaagcttg
60ctttccttga tgacgactgg cgcacgggtg agtaacgcgt atccaacctt cccataacta
120cgggataacc cgttgaaaga cggcctaata ccgtatgata tcgtttgctg
acatcaaata 180acgattaaag gtttagcggt tatggatggg gatgcgtctg
attagcttgt tggcggggta 240acggcccacc aaggctacga tcagtagggg
ttctgagagg aaggtccccc acattggaac 300tgagacacgg tccaaactcc
tacgggaggc agcagtgagg aatattggtc aatgggcgag 360agcctgaacc
agccaagtag cgtgcaggat gacggcccta tgggttgtaa actgctttta
420tgtggggata aagtgcgtga cgtgtcatgc attgcaggta ccacatgaat
aaggaccggc 480taattccgtg 4907490DNAPrevotella OTU47 7gatgaacgct
agctacaggc ttaacacatg caagttgcag ggtaacatga gagaagcttg 60cttttcttga
tgacgactgg cgcacgggtg agtaacgcgt atccaacctt cccataacta
120cgggataacc cgttgaaaga cggcctaata ccgtataaca tcgtttgcag
acatctaacg 180acgattaaag gtttagcggt tatggatggg gatgcgtctg
attagcttgt tggcggggta 240acggcccacc aaggcgacga tcagtagggg
ttctgagagg aaggtccccc acattggaac 300tgagacacgg tccaaactcc
tacgggaggc agcagtgagg aatattggtc aatgggcgag 360agcctgaacc
agccaagtag cgtgtaggat gacggcccta tgggttgtaa actacttttg
420tgtggggata aagtgaggca cgtgtgcctt attgcaggta ccacacgaat
aaggaccggc 480taattccgtg 4908514DNADialister OTU30 8gacgaacgct
ggcggcgtgc ttaacacatg caagtcgaac gggaagacat gaagagcttg 60ctctttatga
aatccagtgg caaacgggtg agtaacacgt aaacaacctg ccttcaggat
120ggggacaaca gacggaaacg actgctaata ccgaatacga ttcttgagtc
gcatgacaca 180agaaagaaag ggtggcctct acttgtaagc tatcgcctgg
agaggggttt gcgtccgatt 240aggtagttgg tgaggtaacg gcccaccaag
ccgacgatcg gtagccggtc tgagaggatg 300aacggccaca ttggaactga
gacacggtcc agactcctac gggaggcagc agtggggaat 360cttccgcaat
gggcgaaagc ctgacggagc aacgccgcgt gagtgaagac ggcctcgggt
420tgtaaactac tgtgattcgg gacgaaggat aagtagacga ataatactgc
ataagtagac 480ggtaccgaaa agacaagacc acggctaaca cgtg
5149513DNADialister OTU167 9gacgaacgct ggcggcgtgc ttaacacatg
caagtcgaac gggaagacat gaagagcttg 60ctctttatga aatccagtgg caaacgggtg
agtaacacgt aaacaacctg ccttcaggat 120ggggacaaca gacggaaacg
actgctaata ccgaatacga ttcttgagtc gcatgacaca 180agaaagaaag
ggtggcctct acaagtaagc tatcgcctga agaggggttt gcgtccgatt
240aggtagttgg tgaggtaacg gcccaccaag ccgacgatcg gtagccggtc
tgagaggatg 300aacggccaca ttggaactga gacacggtcc agactcctac
gggaggcagc agtggggaat 360cttccgcaat gggcgaaagc ctgacggagc
aacgccgcgt gagtgaagac ggccttcggg 420ttgtaaaact ctgtgattcg
ggacgaaaga taagcagacg aataatctgc ataagtgacg 480gtaccgaaaa
agcaagccac ggctaactac gtg 51310490DNAPrevotella amnii 10gatgaacgct
agctataggc ttaacacatg caagtcgagg ggcagcatat agattgcttg 60caatttatga
tggcgaccgg cgcacgggtg agtaacgcgt atccaaccta cccattacta
120gggaataacc cagcgaaagt tggcctaatg ccctatgtag tcgtttgatc
gcctgagatt 180tcgacgaaag atttatcggt attggatggg gatgcgtctg
attagcttgt tggcggggta 240aaggcccacc aaggcgacga tcagtagggg
ttctgagagg aaggtccccc acattggaac 300tgagacacgg tccaaactcc
tacgggaggc agcagtgagg aatattggtc aatgggcgag 360agcctgaacc
agccaagtag cgtgcaggat gacggcccta tgggttgtaa actgctttta
420tatgggaata aagtgaggga cgtgtccctt attgcatgta ccatatgaat
aaggaccggc 480taattccgtg 49011466DNASneathia sanguinegens
11gataaacgct gacagaatgc ttaacacatg caagtcgatg atgggagcta gcttgctaga
60agaagtcatg gcggacgggt gagtaacgtg taaagaactt accatataga ctgggataac
120agagggaaac ttctgataat actggataag ttagtagtag cattactaag
taatgaaagg 180tagcaatacg ctatatgaga gctttgcatc ctattagcta
gttggtgggg taaaagccta 240ccaaggcgat gataggtagc cggcctgaga
gggtggacgg ccacaagggg actgagatac 300ggcccttact cctacgggag
gcagcagtgg ggaatattgg acaatggagg caactctgat 360ccagcaattc
tgtgtgtgtg aagaaggttt taggactgta aaacacattt tagtagggaa
420gaaagaaatg acggtaccta cagaagaagc gacggctaaa tacgtg
46612497DNAAerococcus christensenii 12gacgaacgct ggcggcgtgc
ctaatacatg caagtcgagc gaacagagaa agtgcttgca 60ctttcaaagt tagcggcgga
cgggtgagta acacgtaagg aacctaccga taagcggggg 120acaacatccg
gaaacgggtg ctaataccgc ataggaagtt tgttcgcatg aacaaactta
180gaaagatggc tctgctatca cttatcgatg gccttgcggt gcattaacta
gttggcgagg 240taacggctca ccaaggtgat gatgcatagc cgacctgaga
gggtaatcgg ccacattggg 300actgagacac ggcccaaact cctacgggag
gcagcagtag ggaatcttcc gcaatggacg 360caagtctgac ggagcaacgc
cgcgtgagtg aagaaggttt tcggatcgta aaactctgtt 420gtaagagaag
aacaaattgt agagtaactg ctacagtctt gacggtatct taccagaaag
480ccacggctaa ctacgtg 49713487DNAClostridales BVAB2 13gatgaacgct
ggcggcgtgc ttaacacatg caagtcgaac ggagttaatt tgaggaagca 60agcttgcttg
aagaattaaa ttaacttagt ggcggacggg cgagtaacac gtgagcaacc
120tgcctcttac aggggaataa caacgggaaa ccgttgctaa taccgcataa
catgttgaaa 180gggcatcctt ttaacatcaa aggagcaatc cggtaagaga
tgggctcgcg tccgattagc 240tagttggtag ggtaacggcc taccaaggcg
acgatcggta gccggactga gaggtcgaac 300ggccgcattg ggactgagac
acggcccaga ctcctacggg aggcagcagt ggggaatatt 360gggcaatggg
cgaaagcctg acccagcaac gccgcgtgag tgatgaaggc cttcgggttg
420taaaactctt tggacaggga cgaagaaagt gacggtacct gtagaacaag
ccacggctaa 480ctacgtg 48714462DNACoriobacteriaceae OTU27
14gatgaacgct ggcggcgtgc ctaacacatg caagtcgaac gattaaagca ccttcgggtg
60tgtatagagt ggcgaacggg tgagtaacac gtgaccaacc tgcctcttac attgggacaa
120ccaaaagaaa ttctggctaa taccaaatac tccgcacata tcacatgatg
tatgcgggaa 180agcttttgcg gtaagagatg gggtcgcggc ccattaggta
gacggcgggg tagaagccca 240ccgtgccgat gatgggtagc cgggttgaga
gaccgaccgg ccacattggg actgagatac 300ggcccagact cctacgggag
gcagcagtgg ggaatattgc gcaatggggg aaaccctgac 360gcagcaacgc
cgcgtgcggg atgaaggcct tcgggttgta aaccgctttc agcagggaag
420acatcgacgg tacctgcaga agaagccccg gctaactacg tg
46215511DNADialister micraerophilus 15gacgaacgct ggcggcgtgc
ttaacacatg caagtcgaac gagaggacat gaaaagcttg 60ctttttatga aatctagtgg
caaacgggtg agtaacacgt aaacaacctg ccttcaagat 120ggggacaaca
gacggaaacg actgctaata ccgaatacga tccgaaagtc gcatgacatt
180tggatgaaag ggtggcctat cgaagaagct atcgcttgaa gaggggtttg
cgtccgatta 240ggtagttggt gaggtaacgg cccaccaagc cgacgatcgg
tagccggtct gagaggatga 300acggccacac tggaactgag acacggtcca
gactcctacg ggaggcagca gtggggaatc 360ttccgcaatg gacgaaagtc
tgacggagca acgccgcgtg agtgaagacg gccttcgggt 420tgtaaagctc
tgtgattcgg gacgaaaggc catatgtgaa taatatatgg aaatgacggt
480accgaaaaag caagccacgg ctaactacgt g 51116509DNAMegasphaera OTU70
type 1 16gacgaacgct ggcggcgtgc ttaacacatg caagtcgaac gagaggacat
gggaagcttg 60cttcctatga aatcgagtgg caaacgggtg agtaacgcgt aaacaacctg
cccttcggat 120ggggataaca gccggaaacg gctgctaata ccgaatacga
tcttttcgtc gcatgacgga 180aagaagaaag gatggcctct atacaaagct
atcgccgaag gaggggtttg cgtctgatta 240gctggttgga ggggtaacgg
cccaacaagg cgatgatcag tagccggtct gagaggatga 300acggccacat
tgggactgag acacggccca gactcctacg ggaggcagca gtggggaatc
360ttccgcaatg gacgaaagtc tgacggagca acgccgcgtg agtgaagaag
gtcttcggat 420tgtaaactct gttatacggg acgaaaagac ggatgccaac
agtatccgtc cgtgacggta 480ccgtaagaga agccacggct aactacgtg
50917480DNAParvimonas OTU142 17gacgaacgct ggcggcgtgc ttaacacatg
caagtcgaac gagagttaga tcgaatgagt 60tttcggacaa gtgagattta acgaaagtgg
cgaacgggtg agtaacacgt gagcaacctg 120ccttacacag ggggatagcc
attggaaacg gtgattaata ccccataaga ccacaatacc 180gcatggtaaa
agggtaaaag ggataccggt gtaagatggg ctcgcgtctg attagctagt
240tggtggggta aaggcctacc aaggcgacga tcagtagccg gtctgagagg
atgaacggcc 300acattggaac tgagacacgg tccaaactcc tacgggaggc
agcagtgggg aatattgcac 360aatgggggaa accctgatgc agcgacgccg
cgtgagcgaa gaaggctttc gagtcgtaaa 420gctctgtcct atgagaagat
aatgacggta tcataggagg aagccctggc taaatacgtg
48018519DNALactobacillus crispatus NCTC 4 18gacgaacgct ggcggcgtgc
ctaatacatg caagtcgagc gagcttgcct agatgaattt 60ggtgcttgca ccagatgaaa
ctagatacaa gcgagcggcg gatgggtgag taacacgtgg 120ggaacctgcc
ccatagtctg ggataccact tggaaacagg tgctaatacc ggataagaac
180actagatcgc atgatcagct tttaaaaggc ggcgtaagct gtcgctatgg
gatggccccg 240cggtgcatta gctagttggt aaggtaaagg cttaccaagg
cgatgatgca tagccgagtt 300gagagactga tcggccacat tgggactgag
acacggccca aactcctacg ggaggcagca 360gtagggaatc ttccacaatg
gacgcaagtc tgatggagca acgccgcgtg agtgaagaag 420gttcggctcg
taaagctctg ttggtagtga agaaggatag aggtagtaac tggcctttat
480ttgacggtaa tcaaccagaa agtcacggct aactacgtg
51919512DNALactobacillus crispatus strain ST1 19gacgaacgct
ggcggcgtgc ctaatacatg caagtcgagc gagcggaact aacagattta 60cttcggtaat
gacgttagga aagcgagcgg cggatgggtg agtaacacgt ggggaacctg
120ccccatagtc tgggatacca cttggaaaca ggtgctaata ccggataaga
aagcagatcg 180catgatcagc ttttaaaagg cggcgtaagc tgtcgctatg
ggatggcccc gcggtgcatt 240agctagttgg taaggtaaag gcttaccaag
gcgatgatgc atagccgagt tgagagactg 300atcggccaca ttgggactga
gacacggccc aaactcctac gggaggcagc agtagggaat 360cttccacaat
ggacgcaagt ctgatggagc aacgccgcgt gagtgaagaa ggttttcgga
420tcgtaaagct ctgttgttgg tgaagaagga tagaggtagt aactggcctt
tatttgacgg 480taatcaacca gaaagtcacg gctaactacg tg
51220512DNALactobacillus acidophilus 20gacgaacgct ggcggcgtgc
ctaatacatg caagtcgagc gagctgaacc aacagattca 60cttcggtgat gacgttggga
acgcgagcgg cggatgggtg agtaacacgt ggggaacctg 120ccccatagtc
tgggatacca cttggaaaca ggtgctaata ccggataaga aagcagatcg
180catgatcagc ttataaaagg cggcgtaagc tgtcgctatg ggatggcccc
gcggtgcatt 240agctagttgg tagggtaacg gcctaccaag gcaatgatgc
atagccgagt tgagagactg 300atcggccaca ttgggactga gacacggccc
aaactcctac gggaggcagc agtagggaat 360cttccacaat ggacgaaagt
ctgatggagc aacgccgcgt gagtgaagaa ggttttcgga 420tcgtaaagct
ctgttgttgg tgaagaagga tagaggtagt aactggcctt tatttgacgg
480taatcaacca gaaagtcacg gctaactacg tg 51221512DNALactobacillus
amylovorus 21gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc gagcggaacc
aacagattta 60cttcggtaat gacgctggga aagcgagcgg cggatgggtg agtaacacgt
ggggaacctg 120cccctaagtc tgggatacca tttggaaaca ggtgctaata
ccggataata aagcagatcg 180catgatcagc ttttgaaagg cggcgtaagc
tgtcgctaag ggatggcccc gcggtgcatt 240agctagttgg taaggtaacg
gcttaccaag gcgacgatgc atagccgagt tgagagactg 300atcggccaca
ttgggactga gacacggccc aaactcctac gggaggcagc agtagggaat
360cttccacaat ggacgcaagt ctgatggagc aacgccgcgt gagtgaagaa
ggttttcgga 420tcgtaaagct ctgttgttgg tgaagaagga tagaggtagt
aactggcctt tatttgacgg 480taatcaacca gaaagtcacg gctaactacg tg
51222512DNALactobacillus gallinarum 22gacgaacgct ggcggcgtgc
ctaatacatg caagtcgagc gagcagaacc agcagattta 60cttcggtaat gacgctgggg
acgcgagcgg cggatgggtg agtaacacgt ggggaacctg 120ccccatagtc
tgggatacca cttggaaaca ggtgctaata ccggataaga aagcagatcg
180catgatcagc ttataaaagg cggcgtaagc tgtcgctatg ggatggcccc
gcggtgcatt 240agctagttgg taaggtaacg gcttaccaag gcgatgatgc
atagccgagt tgagagactg 300atcggccaca ttgggactga gacacggccc
aaactcctac gggaggcagc agtagggaat 360cttccacaat ggacgcaagt
ctgatggagc aacgccgcgt gagtgaagaa ggttttcaga 420tcgtaaagct
ctgttgttgg tgaagaagga tagaggtagt aactggcctt tatttgacgg
480taatcaacca gaaagtcacg gctaactacg tg 51223512DNALactobacillus
helveticus 23gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc gagcagaacc
agcagattta 60cttcggtaat gacgctgggg acgcgagcgg cggatgggtg agtaacacgt
ggggaacctg 120ccccatagtc tgggatacca cttggaaaca ggtgctaata
ccggataaga aagcagatcg 180catgatcagc ttataaaagg cggcgtaagc
tgtcgctatg ggatggcccc gcggtgcatt 240agctagttgg taaggtaacg
gcttaccaag gcaatgatgc atagccgagt tgagagactg 300atcggccaca
ttgggactga gacacggccc aaactcctac gggaggcagc agtagggaat
360cttccacaat ggacgcaagt ctgatggagc aacgccgcgt gagtgaagaa
ggttttcgga 420tcgtaaagct ctgttgttgg tgaagaagga tagaggtagt
aactggcctt tatttgacgg 480taatcaacca gaaagtcacg gctaactacg tg
51224512DNALactobacillus kitasatonis 24gacgaacgct ggcggcgtgc
ctaatacatg caagtcgagc gagcggaacc aacagattta 60cttcggtaat gacgttggga
aagcgagcgg cggatgggtg agtaacacgt ggggaacctg 120cccctaagtc
tgggatacca tttggaaaca ggtgctaata ccggataaga aagcagatcg
180catgatcagc ttttaaaagg cggcgtaagc tgtcgctaag ggatggcccc
gcggtgcatt 240agctagttgg taaggtaacg gcttaccaag gcaacgatgc
atagccgagt tgagagactg 300atcggccaca ttgggactga gacacggccc
aaactcctac gggaggcagc agtagggaat 360cttccacaat ggacgcaagt
ctgatggagc aacgccgcgt gagtgaagaa ggttttcgga 420tcgtaaagct
ctgttgttgg tgaagaagga tagaggtagt aactggcctt tatttgacgg
480taatcaacca gaaagtcacg gctaactacg tg 51225512DNALactobacillus
sobrius 25gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc gagcggaacc
aacagattta 60cttcggtaat gacgttggga aagcgagcgg cggatgggtg agtaacacgt
ggggaacctg 120cccctaagtc tgggatacca tttggaaaca ggtgctaata
ccggataata aagcagatcg 180catgatcagc ttttgaaagg cggcgtaagc
tgtcgctaag ggatggcccc gcggtgcatt 240agctagttgg taaggtaacg
gcttaccaag gcgacgatgc atagccgagt tgagagactg 300atcggccaca
ttgggactga gacacggccc aaactcctac gggaggcagc agtagggaat
360cttccacaat ggacgcaagt ctgatggagc aacgccgcgt gagtgaagaa
ggttttcgga 420tcgtaaagct ctgttgttgg tgaagaagga tagaggtagt
aactggcctt tatttgacgg 480taatcaacca gaaagtcacg gctaactacg tg
51226512DNALactobacillus ultunensis 26gacgaacgct ggcggcgtgc
ctaatacatg caagtcgagc gagcggaacc agcagatctg 60cttcggcagt gacgctggga
aagcgagcgg cggatgggtg agtaacacgt ggggaacctg 120ccccaaagtc
tgggatacca cttggaaaca ggtgctaata ccggataaga aagcagatcg
180catgatcagc ttttaaaagg cggcgtaagc tgtcgctatg ggatggcccc
gcggtgcatt 240agctagttgg tagagtaacg gcctaccaag gcaatgatgc
atagccgagt tgagagactg 300atcggccaca ttgggactga gacacggccc
aaactcctac gggaggcagc agtagggaat 360cttccacaat ggacgcaagt
ctgatggagc aacgccgcgt gagtgaagaa ggttttcgga 420tcgtaaagct
ctgttgttgg tgaagaagga tagaggtagt aactggcctt tatttgacgg
480taatcaacca gaaagtcacg gctaactacg tg 512
* * * * *
References